共查询到20条相似文献,搜索用时 31 毫秒
1.
Kyungsoon Kim 《Biotechnology and Bioprocess Engineering》2002,7(4):216-220
1,4-Benzoquinone reductase was purified to electrophoretic homogeneity from bovine liver, and the purified enzyme found to
have a molecular mass of 29 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited
pH optimum between 8.0 and 8.5. The apparentK
m for 1,4-benzoquinone was 1.643 mM, and the apparent Km for NADH was 1.837 mM. Various divalent cations, such as Hg2+, Cu2+, and Zn2+, exhibited strong inhibitory effects. The enzyme activity was also strongly inhibited by quercetin, dicumarol, and benzoic
acid. Incubation of the enzyme withN-bromosuccinimide and pyridoxal 5′-phosphate led to inhibitions of 100% and 99%, respectively. Accordingly, these results
suggest that tryptophan and lysine residues are involved at or near the active sites of the enzyme. 相似文献
2.
The gene encoding a cold-active and xylose-stimulated β-glucosidase of Marinomonas MWYL1 was synthesized and expressed in Escherichia coli. The recombinant enzyme (reBglM1) was purified and characterized. The molecular mass of the purified reBglM1 determined by
SDS-PAGE agree with the calculated values (50.6 Da). Optima of temperature and pH for enzyme activity were 40°C and 7.0, respectively.
The enzyme exhibited about 20% activity at 5°C and was stable over the range of pH 5.5–10.0. The presence of xylose significantly
enhanced enzyme activity even at higher concentrations up to 600 mM, with maximal stimulatory effect (about 1.45-fold) around
300 mM. The enzyme is active with both glucosides and galactosides and showed high catalytic efficiency (k
cat = 500.5 s−1) for oNPGlc. These characterizations enable the enzyme to be a promising candidate for industrial applications. 相似文献
3.
Purification and characterization of an ADP-dependent phosphofructokinase from Thermococcus zilligii 总被引:1,自引:0,他引:1
R. S. Ronimus Jurre Koning H. W. Morgan 《Extremophiles : life under extreme conditions》1999,3(2):121-129
The ADP-dependent phosphofructokinase (PFK) from Thermococcus zilligii has been purified 950 fold; it had a specific activity of 190 U mg−1. The enzyme required Mg2+ ions for optimal activity and was specific for ADP. The forward reaction kinetics were hyperbolic for both cosubstrates (pH
optimum of 6.4), and the apparent K
m values for ADP and fructose-6-phosphate were 0.6 mM (apparent V
max of 243 U mg−1) and 1.47 mM (apparent V
max of 197 U mg−1), respectively. Significantly, the enzyme is indicated to be nonallosteric but was slightly activated by some monovalent
cations including Na+ and K+. The protein had a subunit size of 42.2 kDa and an estimated native molecular weight of 66 kDa (gel filtration). Maximal
reaction rates for the reverse reaction were attained at pH 7.5–8.0, and the apparent K
m values for fructose-1,6-bisphosphate and AMP were 0.56 mM (apparent V
max of 2.9 U mg−1) and 12.5 mM, respectively. The biochemical characteristics of this unique ADP-dependent enzymatic activity are compared
to ATP and pyrophosphate-dependent phosphofructokinases.
Received: August 14, 1998 / Accepted: December 2, 1998 相似文献
4.
A thermophilic Bacillus sp. was isolated that secreted an extracellular, thermostable lipolytic enzyme. The enzyme was purified to 58 folds with
a specific activity of 9730 units/mg of protein and yield of 10% activity by ammonium sulphate precipitation, Phenyl Sepharose
chromatography, gel-permeation followed by Q Sepharose chromatography. The relative molecular mass of the protein was determined
to be 61 kDa by SDS-PAGE and approximately 60 kDa by gel permeation chromatography. The enzyme showed optimal activity at
60–65 ∘C and retained 100% activity after incubation at 60 ∘C and pH 8.0 for 1 h. The optimum pH was determined to be 8.5. It exhibited 50% of its original activity after 65 min incubation
at 70 ∘C and 23 min incubation at 80 ∘C. Catalytic function of lipase was activated by Mg++ (10 mM), while mercury (10 mM) inactivated the enzyme completely. No effect on enzyme activity was observed with trypsin
and chymotrypsin treatment, while 50% inhibition was observed with thermolysin. It was demonstrated that PMSF, SDS, DTT, EDTA,
DEPC, βME (100 mM each) and eserine (10 mM) inhibited the activity of the lipolytic enzyme. With p-nitrophenyl laurate as
a substrate, the enzyme exhibited a K
m
and V
max of 0.5 mM and 0.139 μM/min/ml. The enzyme showed preference for short chain triacylglycerol and hydrolyzes triolein at all
positions. In contrast to other thermostable Bacillus lipases, this enzyme has very low content of hydrophobic amino acids (22.58 %). Immunological studies showed that the active
site and antigen-binding site of enzyme do not overlap. 相似文献
5.
When challenged with reactive oxidants, the nonsulfur phototrophic bacterium Rhodobacter sphaeroides ATH 2.4.1 exhibited an oxidative stress response during both phototrophic and chemotrophic growth. Upon preincubation with
100 μM H2O2, catalase activity increased fivefold. Catalase was also induced by other forms of oxidative stress, heat-shock, ethanol
treatment, and stationary-phase conditions. Only one band of catalase activity was detected after native and denaturing PAGE.
The enzyme was purified 304-fold with a yield of 7%. The purified enzyme displayed a heterodimeric structure with subunits
of 75 and 68 kDa, corresponding to a molecular mass of approximately 150 kDa for the native enzyme. The subunits had almost
identical amino-terminal peptide sequences, sharing substantial similarity with other bacterial catalases. The enzyme exhibited
an apparent K
m of 40 mM and a V
max of 285,000 U (mg protein)–1. Spectroscopic analysis indicated the presence of protoheme IX. The heme content calculated from pyridine hemochrome spectra
was 0.43 mol per mol of enzyme. The enzyme had a broad pH optimum and was inhibited by cyanide, azide, hydroxylamine, 2-mercaptoethanol,
and sodium dithionite. These data indicate that this catalase belongs to the class of monofunctional catalases.
Received: 15 October 1997 / Accepted: 2 February 1998 相似文献
6.
F. -M. Zhu B. Du H. -S. Gao C. -J. Liu J. Li 《Applied Biochemistry and Microbiology》2010,46(6):626-632
Protoplasts of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 were prepared using cellulose and snail enzyme with 0.6 M NaCl as osmotic stabilizer. Protoplast fusion has been performed
using 35% polyethylene glycol 4,000 with 0.01 mM CaCl2. The fused protoplasts have been regenerated on regeneration medium and fusants were selected for further studies. An intracellular
(β-glucosidase (EC 3.2.1.21) was purified from the protoplast fusant of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 and characterized. The enzyme was purified 138.85-fold by ammonium sulphate precipitation, DE-22 ion exchange and Sephadex
G-150 gel filtration chromatography with a specific activity of 297.14 U/mg of protein. The molecular mass of the purified
enzyme was determined to be about 125 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme
had an optimum pH of 5.4 and temperature of 65°C, respectively. This enzyme showed relatively high stability against pH and
temperature and was stable in the pH range of 3.0–6.6. Na+, K+, Ca2+, Mg2+ and EDTA completely inhibited the enzyme activity at a concentration of 10 mM. The enzyme activity was accelerated by Fe3+. The enzyme activity was strongly inhibited by glucose, the end product of glucoside hydrolysis. The K
m and V
max values against salicin as substrate were 0.035 mM and 1.7215 μmol min−1, respectively. 相似文献
7.
An acyl-coenzyme A carboxylase that carboxylates acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA was purified from
Myxococcus xanthus. Since the enzyme showed maximal rates of carboxylation with propionyl-CoA, the enzyme is thought to be propionyl-CoA carboxylase.
The apparent K
m values for acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA were found to be 0.2, 0.2, 0.03, and 1.0 mM, respectively.
The native enzyme has a molecular mass of 605–615 kDa and is composed of nonidentical subunits (α and β) with molecular masses
of 53 and 56 kDa, respectively. The enzyme showed maximal activity at pH 7.0–7.5 and at 25–30°C, and was affected by variation
in concentrations of ATP and Mg2+. During development of M. xanthus, the propionyl-CoA carboxylase activity increased gradually, with maximum activity observed during the sporulation stage.
Previous work has shown that a propionyl-CoA-carboxylase-deficient mutant of M. xanthus reduces levels of long-chain fatty acids. These results suggest that the propionyl-CoA carboxylase is also responsible for
the carboxylation of acetyl-CoA to malonyl-CoA used for the synthesis of long-chain fatty acids during development.
Received: 24 February 1998 / Accepted: 25 May 1998 相似文献
8.
Cathespin B has been purified 750-fold to apparent homogeneity from human and bovine brain cortex using ammonium sulfate fractionation
(30–70%), chromatography on Sephadex G-100, CM-Sephadex C-50, and concanavalin A-Sepharose. Enzyme was assayed fluorometrically
at pH 4.0 with pyridoxyl-hemoglobin in the presence of 1 mM DTT and 1 mM EDTA. Properties of the enzyme from the two sources
proved to be similar. On disc PAGE the purified preparation produced two bands associated with proteinase activity that are
due to existence of two multiple forms of brain cathepsin B with pI 6.1 and 6.8. The enzyme is completely inactivated by thiol-blocking
reagents, leupeptin, E-64, and demands thiol compounds for its ultimate activity. Z-Phe-Ala-CHN2 is a potent inhibitor of the enzyme (K
2nd=1280 M−1s−1) in contrast to Z-Phe-Phe-CHN2 (K
2nd=264 M−1s−1). pH optimum in the reaction of hydrolysis of Pxy-Hb is 4.0–6.0,K
M(app.) =10−5 M. Cathepsin B splits azocasein: pH optimum 5.0–6.0,K
M(app.)=2.2·10−5 M, but inclusion of urea in the incubation medium depresses the azocaseinolytic activity of the enzyme 1.5-fold. It does
not split Lys-NNap, Arg-NMec and is not inhibited by bestatin. The specific activity of brain cathepsin B with Z-Arg-Arg-NNapOMe
at pH 6.0 is 10-fold higher than with Bz-Arg-NNap, Z-Gly-Gly-Arg-NNap is a poor substrate. With Z-Arg-Arg-NMec and Bz-Phe-Val-Arg-NMec
the specific acitivity is 80 and 35%, respectively of that with Z-Phe-Arg-NMec.
Special Issue dedicated to Dr. Eugene Kreps. 相似文献
9.
B B Nepple J Kessi R Bachofen 《Journal of industrial microbiology & biotechnology》2000,25(4):198-203
Rhodobacter sphaeroides grew in the presence of up to 43 μM chromate and reduced hexavalent chromium to the trivalent form under both aerobic and
anaerobic conditions. Reduced chromium remained in the external medium. Reductase activity was present in cells of R. sphaeroides independent of whether chromate was present or not in the growth medium. The reducing activity was found in the cytoplasmic
cell fraction and was dependent on NADH. The chromate-reducing enzyme was purified by anion exchange, hydroxyapatite and hydrophobic
interaction chromatography, and gel filtration. The molecular weight of the enzyme was 42 kDa as determined by gel filtration.
The optimum of the reaction is at pH 7.0 and 30°C. The enzyme activity showed a hyperbolic dependence on the concentrations
of both substrates, NADH and chromate, with a maximum velocity at 0.15 mM NADH. A K
m of 15±1.3 μM CrO4
2− and a V
max of 420±50 μmol min−1 mg protein−1 was determined for the enzyme isolated from anaerobically grown cells and 29±6.4 μM CrO4
2− and 100±9.6 μmol CrO4
2− min−1 mg protein−1 for the one from aerobically grown ones. Journal of Industrial Microbiology & Biotechnology (2000) 25, 198–203.
Received 05 January 2000/ Accepted in revised form 27 May 2000 相似文献
10.
Hirasawa K Uchimura K Kashiwa M Grant WD Ito S Kobayashi T Horikoshi K 《Antonie van Leeuwenhoek》2006,89(2):211-219
An endoglucanase was purified to homogeneity from an alkaline culture broth of a strain isolated from␣seawater and identified
here as Bacillus agaradhaerens JAM-KU023. The molecular mass was around 38-kDa and the N-terminal 19 amino acids of the purified enzyme exhibited 100% sequence
identity to Cel5A of B. agaradhaerens DSM8721T. The enzyme activity increased around 4-fold by the addition of 0.2–2.0 M NaCl in 0.1 M glycine–NaOH buffer (pH 9.0). KCl,
Na2SO4, NaBr, NaNO3, CH3COONa, LiCl, NH4NO3, and NH4Cl also activated the enzyme up to 2- to 4-fold. The optimal pH and temperature values were pH 7–9.4 and 60 °C with 0.2 M
NaCl, but pH 6.5–7 and 50 °C without NaCl; enzyme activity increased approximately 6-fold at 60 °C with 0.2 M NaCl compared
to that at 50 °C without NaCl in 0.1 M glycine–NaOH buffer (pH 9.0). The thermostability and pH stability of the enzyme were
not affected by NaCl. The enzyme was very stable to several chemical compounds, surfactants and metal ions (except for Fe2+ and Hg2+ ions), regardless whether NaCl was present or not.
* The nucleotide sequence of 16S rRNA of this strain has been submitted to DDBJ, EMBL, and GenBank databases under accession
no. AB211544. 相似文献
11.
Jin Zhou Ju Chu Yong-Hong Wang Si-Liang Zhang Ying-Ping Zhuang Zhong-Yi Yuan 《World journal of microbiology & biotechnology》2008,24(6):789-796
An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH4)2SO4 fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed
the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular
weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric
point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5
and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature
stability was up to 45 °C. Metal ions, such as, Mn2+ and K+ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg2+ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu2+ and Ag2+) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K
m of 120 and 330 μM and V
max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively. 相似文献
12.
Sumra Afzal Mahjabeen Saleem Riffat Yasmin Mamoona Naz Muhammad Imran 《Molecular biology reports》2010,37(4):1717-1723
Consistent with its precloning characterization from the cellulolytic Bacillus sp., β-1,4-endoglucanase purified from the recombinant E. coli exhibited maximum activity at 60°C and pH 7.0. It was highly specific for CMC hydrolysis, with stability up to 70°C and over
a pH range of 6.0–8.0. The K
m and V
max values for CMCase activity of the enzyme were 4.1 mg/ml and 25 μmole/ml min−1, respectively. The purified enzyme was a monomer of 65 kDa, as determined by SDS-PAGE. The presence of sucrose and IPTG in
fermentation media increased the endoglucanase activity of the recombinant enzyme to 5.2-folds as compared with that of the
actual one. 相似文献
13.
The invertase of Lactobacillus reuteri CRL 1100 is a glycoprotein composed by a single subunit with a molecular weight of 58 kDa. The enzyme was stable below 45°C
over a wide pH range (4.5–7.0) with maximum activity at pH 6.0 and 37°C. The invertase activity was significantly inhibited
by bivalent metal ions (Ca++, Cu++, Cd++, and Hg++), β-mercaptoethanol, and dithiothreitol and partially improved by ethylenediaminetetraacetic acid. The enzyme was purified
32 times over the crude extract by gel filtration and ion-exchange chromatography with a recovery of 17%. The K
m
and Vmax values for sucrose were 6.66 mM and 0.028 μmol/min, respectively. An invertase is purified and characterized for the first
time in Lactobacillus, and it proved to be a β-fructofuranosidase.
Received: 13 August 1999 / Accepted: 15 September 1999 相似文献
14.
El-Sayed AS 《Journal of microbiology (Seoul, Korea)》2011,49(1):130-140
L-Methioninase was purified to electrophoretic homogeneity from cultures of Aspergillus flavipes using anion-exchange and gel filtration chromatography by 12.1 fold compared to the crude enzyme preparation. The purified
enzyme had a molecular mass of 47 kDa under denaturing conditions and an isoelectric point of 5.8 with no structural glycosyl
residues. The enzyme had optimum activity at pH 7.8 and pH stability from 6.8–8.0 at 35°C. The enzyme appeared to be catalytically
stable below 40°C. The enzyme activity was strongly inhibited by DL-propargylglycine, hydroxylamine, PMSF, 2-mercaptoethanol,
Hg+, Cu2+, and Fe2+, with slight inhibition by Triton X-100. A flavipes L-methioninase has a higher catalytic affinity towards L-methionine (Km, 6.5 mM and Kcat, 14.1 S−1) followed by a relative demethiolating activity to L-homo-cysteine (Km, 12 mM and Kcat, 9.3 S−1). The enzyme has two absorption maxima at 280 and 420 nm, typical of other PLP-enzymes. Apo-L-methioninase has the ability
to reconstitute its structural catalytic state completely upon addition of 0.15 mM PLP. L-Methioninase has neither an appreciable
effect on liver function, platelet aggregation, nor hemolysis of human blood. The purified L-methioninase from solid cultures
of A. flavipes displayed unique biochemical and catalytic properties over the currently applied Pseudomonad enzyme. 相似文献
15.
A soluble glucoside 3-dehydrogenase (G3DH) from Stenotrophomonas maltrophilia CCTCC M 204024, recently isolated from wheat soil in our laboratory, was purified to 37.4-fold with a yield of 24.7% and was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 66 kDa. 2,6-Dichlorophenolindophenol (DCPIP) and ferricyanide were able to act as artificial electron acceptors for the enzyme. The optimal pH of G3DH was in the range of 6.0–7.0 in the presence of DCPIP. The enzyme was stable in the pH range of 4.4–10.6 and was sensitive to heat. G3DH exhibited extremely broad substrate specificity by converting many sugars to their corresponding 3-ketoglucosides. They produced a characteristic spectrum by alkaline treatment with a peak at 340 nm. The apparent K
m values for validoxylamine A and d-glucose were 8.3 and 1.1 mM, respectively. Cu2+, Ag2+, and Hg2Cl2 inhibited the activity of G3DH. 相似文献
16.
NAD+-linked primary and secondary alcohol dehydrogenase activity was detected in cell-free extracts of propane-grown Rhodococcus rhodochrous PNKb1. One enzyme was purified to homogeneity using a two-step procedure involving DEAE-cellulose and NAD-agarose chromatography and this exhibited both primary and secondary NAD+-linked alcohol dehydrogenase activity. The Mr of the enzyme was approximately 86,000 with subunits of Mr 42,000. The enzyme exhibited broad substrate specificity, oxidizing a range of short-chain primary and secondary alcohols (C2–C8) and representative cyclic and aromatic alcohols. The pH optimum was 10. At pH 6.5, in the presence of NADH, the enzyme catalysed the reduction of ketones to alcohols. The K
m values for propan-1-ol, propan-2-ol and NAD were 12 mM, 18 mM and 0.057 mM respectively. The enzyme was inhibited by metal-complexing agents and iodoacetate. The properties of this enzyme were compared with similar enzymes in the current literature, and were found to be significantly different from those thus far described. It is likely that this enzyme plays a major role in the assimilation of propane by R. rhodochrous PNKb1.Abbreviations HPLC
high performance liquid chromatography
- DEAE
diethyl amino ethyl
- IEF
isoelectrofocusing
- NTG
nitrosoguanidine
- SDS-PAGE
sodium dodecylsulphate polyacrylamide gel electrophoresis
- pI
isoelectric point 相似文献
17.
Miake F Satho T Takesue H Yanagida F Kashige N Watanabe K 《Archives of microbiology》2000,173(1):65-70
α-l-Rhamnosidase was extracted and purified from the cells of Pseudomonas paucimobilis FP2001 with a 19.5% yield. The purified enzyme, which was homogeneous as shown by SDS-PAGE and isoelectric focusing, had
a molecular weight of 112,000 and an isoelectric point of 7.1. The enzyme activity was accelerated by Ca2+ and remained stable for several months when stored at –20 °C. The optimum pH was 7.8; the optimum temperature was 45 °C.
The K
m, V
max and k
cat for p-nitrophenyl α-l-rhamnopyranoside were 1.18 mM, 92.4 μM · min–1 and 117,000 · min–1, respectively. Examination of the substrate specificity using various synthetic and natural l-rhamnosyl glycosides showed that this enzyme had a relatively broader substrate specificity than those reported so far.
Received: 24 May 1999 / Accepted: 7 October 1999 相似文献
18.
NADH-dependent NO scavenging in barley extracts is linked to hemoglobin (Hb) expression and is inhibited by SH-reagents. Barley
Hb has a single cysteine residue. To determine whether this cysteine was critical for NO scavenging, barley Hb and a mutated
version, in which the single Cys79 was replaced by Ser, were over-expressed in Escherichia coli and purified to near homogeneity. The purified proteins exhibited very low NO-scavenging activity (12–14 nmol min−1 mg−1 protein) in the presence of NADH or NADPH. This activity was insensitive to SH-reagents. Addition of an extract from barley
roots to either of the purified proteins resulted in high NADH-dependent NO turnover in a reaction that was sensitive to SH-reagents.
A protein was purified from barley roots and identified by mass-spectrometry analysis as a cytosolic monodehydroascorbate
reductase. It efficiently supported NADH-dependent NO scavenging in the presence of either native or mutated barley Hb. Ascorbate
strongly facilitated the rate of metHb reduction. The K
m for Hb was 0.3 μM, for ascorbate 0.6 mM and for NADH 4 μM. The reaction in the presence of monodehydroascorbate reductase
was sensitive to SH-reagents with either form of the Hb. We conclude that metHb reduction and NO turnover do not involve direct
participation of the Cys79 residue of barley Hb. NO scavenging is facilitated by monodehydroascorbate reductase mediating a coupled reaction involving
ferric Hb reduction in the presence of ascorbate and NADH. 相似文献
19.
Sanjay Chauhan Anuradha Nichkawade M.R.S. Iyengar Bharat B. Chattoo 《Current microbiology》1998,37(3):186-190
Aerobic cultures of an actinomycete were found to produce penicillin V acylase (PVA) (PA, EC-3.5.1.11) extracellularly. The
presence of L-2-3 diamino-propionic acid in cell wall and formation of sclerotia on culture media led to its identification
as Chainia, a sclerotial Streptomyces. Partially purified acylase was adsorbed on kieselguhr and entrapped in polyacrylamide gel. The immobilized preparation proved
effective with respect to retention of enzyme and enzyme activity even after 15 successful cycles. The pH optimum for crude
enzyme was in the range of pH 7.5–8.0, and for the (NH4)2 SO4 fraction it was pH 8.5. The immobilized enzyme showed maximal activity at pH 9.5. The optimum temperature for acylase activity
was at 55°C. The crude enzyme, ammonium sulfate fraction, and immobilized enzyme showed K
m
value for penicillin V of 6.13 mM, 14.3 mM, and 17.1 mM, respectively.
Received: 11 December 1997 / Accepted: 9 April 1998 相似文献
20.
S Chakraborti R K Sani U C Banerjee R C Sobti 《Journal of industrial microbiology & biotechnology》2000,24(1):58-63
An extracellular β-galactosidase which catalyzed the production of galacto-oligosaccharide from lactose was harvested from
the late stationary-phase of Bacillus sp MTCC 3088. The enzyme was purified 36.2-fold by ZnCl2 precipitation, ion exchange, hydrophobic interaction and gel filtration chromatography with an overall recovery of 12.7%.
The molecular mass of the purified enzyme was estimated to be about 484 kDa by gel filtration on a Sephadex G-200 packed column
and the molecular masses of the subunits were estimated to be 115, 86.5, 72.5, 45.7 and 41.2 kDa by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis. The isoelectric point of the native enzyme, determined by polyacrylamide gel electrofocusing, was 6.2.
The optimum pH and temperature were 8 and 60°C, respectively. The Michaelis–Menten constants determined with respect to o-NO2-phenyl-β-D-galactopyranoside and lactose were 6.34 and 6.18 mM, respectively. The enzyme activity was strongly inhibited (68%) by galactose,
the end product of lactose hydrolysis reaction. The β-galactosidase was specific for β-D anomeric linkages. Enzyme activity was significantly inhibited by metal ions (Hg2+, Cu2+ and Ag+) in the 1–2.5 mM range. Mg2+ was a good activator. Catalytic activity was not affected by the chelating agent EDTA. Journal of Industrial Microbiology & Biotechnology (2000) 24, 58–63.
Received 09 February 1999/ Accepted in revised form 24 September 1999 相似文献