Biochemical and phylogenetic analyses of a cold-active beta-galactosidase from the lactic acid bacterium Carnobacterium piscicola BA

We are investigating glycosyl hydrolases from new psychrophilic isolates to examine the adaptations of enzymes to low temperatures. A beta-galactosidase from isolate BA, which we have classified as a strain of the lactic acid bacterium Carnobacterium piscicola, was capable of hydrolyzing the chromogen 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-Gal) at 4 degrees C and possessed higher activity in crude cell lysates at 25 than at 37 degrees C. Sequence analysis of a cloned DNA fragment encoding this activity revealed a gene cluster containing three glycosyl hydrolases with homology to an alpha-galactosidase and two beta-galactosidases. The larger of the two beta-galactosidase genes, bgaB, encoded the 76.8-kDa cold-active enzyme. This gene was homologous to family 42 glycosyl hydrolases, a group which contains several thermophilic enzymes but none from lactic acid bacteria. The bgaB gene from isolate BA was subcloned in Escherichia coli, and its enzyme, BgaB, was purified. The purified enzyme was highly unstable and required 10% glycerol to maintain activity. Its optimal temperature for activity was 30 degrees C, and it was inactivated at 40 degrees C in 10 min. The K(m) of freshly purified enzyme at 30 degrees C was 1.7 mM, and the V(max) was 450 micromol. min(-1). mg(-1) with o-nitrophenyl beta-D-galactopyranoside. This cold-active enzyme is interesting because it is homologous to a thermophilic enzyme from Bacillus stearothermophilus, and comparisons could provide information about structural features important for activity at low temperatures.     

Characterization of two new glycosyl hydrolases from the lactic acid bacterium Carnobacterium piscicola strain BA.

Three genes with homology to glycosyl hydrolases were detected on a DNA fragment cloned from a psychrophilic lactic acid bacterium isolate, Carnobacterium piscicola strain BA. A 2.2-kb region corresponding to an alpha-galactosidase gene, agaA, was followed by two genes in the same orientation, bgaB, encoding a 2-kb beta-galactosidase, and bgaC, encoding a structurally distinct 1.76-kb beta-galactosidase. This gene arrangement had not been observed in other lactic acid bacteria, including Lactococcus lactis, for which the genome sequence is known. To determine if these sequences encoded enzymes with alpha- and beta-galactosidase activities, we subcloned the genes and examined the enzyme properties. The alpha-galactosidase, AgaA, hydrolyzes para-nitrophenyl-alpha-D-galactopyranoside and has optimal activity at 32 to 37 degrees C. The beta-galactosidase, BgaC, has an optimal activity at 40 degrees C and a half-life of 15 min at 45 degrees C. The regulation of these enzymes was tested in C. piscicola strain BA and activity on both alpha- and beta-galactoside substrates decreased for cells grown with added glucose or lactose. Instead, an increase in activity on a phosphorylated beta-galactoside substrate was found for the cells supplemented with lactose, suggesting that a phospho-galactosidase functions during lactose utilization. Thus, the two beta-galactosidases may act synergistically with the alpha-galactosidase to degrade other polysaccharides available in the environment.     

β-Galactosidase from a cold-adapted bacterium: purification, characterization and application for lactose hydrolysis

The enzyme beta-galactosidase was purified from a cold-adapted organism isolated from Antarctica. The organism was identified as a psychotrophic Pseudoalteromonas sp. The enzyme was purified with high yields by a rapid purification scheme involving extraction in an aqueous two-phase system followed by hydrophobic interaction chromatography and ultrafiltration. The beta-galactosidase was optimally active at pH 9 and at 26 degrees C when assayed with o-nitrophenyl-beta-D-galactopyranoside as substrate for 2 min. The enzyme activity was highly sensitive to temperature above 30 degrees C and was undetectable at 40 degrees C. The cations Na+, K+, Mg2+ and Mn2+ activated the enzyme while Ca2+, Hg2+, Cu2+ and Zn2+ inhibited activity. The shelf life of the pure enzyme at 4 degrees C was significantly enhanced in the presence of 0.1% (w/v) polyethyleneimine. The pure beta-galactosidase was also evaluated for lactose hydrolysis. More than 50% lactose hydrolysis was achieved in 8 h in buffer at an enzyme concentration of 1 U/ml, and was increased to 70% in the presence of 0.1% (w/v) polyethyleneimine. The extent of lactose hydrolysis was 40-50% in milk. The enzyme could be immobilized to Sepharose via different chemistries with 60-70% retention of activity. The immobilized enzyme was more stable and its ability to hydrolyze lactose was similar to that of the soluble enzyme.     

Purification and characterization of a beta-galactosidase from peach (Prunus persica)

A beta-galactosidase (EC 3.2.1.23) from peach (Prunus persica cv Mibackdo) was purified and characterized. The purified peach beta-galactosidase was 42 kDa in molecular mass and showed high enzyme activity against a the beta-galactosidase substrate, rho-nitrophenyl-beta-D-galactopyranoside. The Km and Vmax values of the enzyme activity of the peach beta-galactosidase were 5.16 and 0.19 mM for rho-nitrophenyl-beta-D-galactopyranoside mM/h, respectively. The optimum pH of the enzyme activity was pH 3.0, but it was relatively stable from pH 3.0-10.0. The temperature optimum was 50 degrees C. The enzyme activities were not improved in the buffers that contained Ca2+, Cu2+, Zn2+, and Mg2+, which indicates that the purified peach beta-galactosidase did not require these cations as co-factors. However, the enzyme was completely inhibited by Hg2+. The purified protein was cross-reacted with an antibody against the persimmon fruit beta-galactosidase. A further comparison of the N-terminal amino acid sequence of the purified protein showed high homologies to those of beta-galactosidase in apple (87%), persimmon (80%), and tomato (87%). Therefore, enzymatic, immunological, and molecular evidences in this study indicate that the purified 42-kDa protein is a peach beta-galactosidase.     

Identification and molecular characterization of the first alpha -xylosidase from an archaeon

We here report the first molecular characterization of an alpha-xylosidase (XylS) from an Archaeon. Sulfolobus solfataricus is able to grow at temperatures higher than 80 degrees C on several carbohydrates at acidic pH. The isolated xylS gene encodes a monomeric enzyme homologous to alpha-glucosidases, alpha-xylosidases, glucoamylases and sucrase-isomaltases of the glycosyl hydrolase family 31. xylS belongs to a cluster of four genes in the S. solfataricus genome, including a beta-glycosidase, an hypothetical membrane protein homologous to the major facilitator superfamily of transporters, and an open reading frame of unknown function. The alpha-xylosidase was overexpressed in Escherichia coli showing optimal activity at 90 degrees C and a half-life at this temperature of 38 h. The purified enzyme follows a retaining mechanism of substrate hydrolysis, showing high hydrolytic activity on the disaccharide isoprimeverose and catalyzing the release of xylose from xyloglucan oligosaccharides. Synergy is observed in the concerted in vitro hydrolysis of xyloglucan oligosaccharides by the alpha-xylosidase and the beta-glycosidase from S. solfataricus. The analysis of the total S. solfataricus RNA revealed that all the genes of the cluster are actively transcribed and that xylS and orf3 genes are cotranscribed.     

Bioinformatic, genetic, and biochemical evidence that some glycoside hydrolase family 42 beta-galactosidases are arabinogalactan type I oligomer hydrolases

Glycoside hydrolases are organized into glycoside hydrolase families (GHFs) and within this larger group, the beta-galactosidases are members of four families: 1, 2, 35, and 42. Most genes encoding GHF 42 enzymes are from prokaryotes unlikely to encounter lactose, suggesting a different substrate for these enzymes. In search of this substrate, we analyzed genes neighboring GHF 42 genes in databases and detected an arrangement implying that these enzymes might hydrolyze oligosaccharides released by GHF 53 enzymes from arabinogalactan type I, a pectic plant polysaccharide. Because Bacillus subtilis has adjacent GHF 42 and GHF 53 genes, we used it to test the hypothesis that a GHF 42 enzyme (LacA) could act on the oligosaccharides released by a GHF 53 enzyme (GalA) from galactan. We cloned these genes, plus a second GHF 42 gene from B. subtilis, yesZ, into Escherichia coli and demonstrated that cells expressing LacA with GalA gained the ability to use galactan as a carbon source. We constructed B. subtilis mutants and showed that the increased beta-galactosidase activity generated in response to the addition of galactan was eliminated by inactivating lacA or galA but unaffected by the inactivation of yesZ. As further demonstration, we overexpressed the LacA and GalA proteins in E. coli and demonstrated that these enzymes degrade galactan in vitro as assayed by thin-layer chromatography. Our work provides the first in vivo evidence for a function of some GHF 42 beta-galactosidases. Similar functions for other beta-galactosidases in both GHFs 2 and 42 are suggested by genomic data.     

A new α-galactosidase from symbiotic Flavobacterium sp. TN17 reveals four residues essential for α-galactosidase activity of gastrointestinal bacteria

The α-galactosidase gene, galA17, was cloned from Flavobacterium sp. TN17, a symbiotic bacterium isolated from the gut of Batocera horsfieldi larvae. The 2,205-bp full-length gene encodes a 734-residue polypeptide (GalA17) containing a putative 28-residue signal peptide and a catalytic domain belonging to glycosyl hydrolase family 36 (GH 36). The deduced amino acid sequence of galA17 was most similar to a putative α-galactosidase from Pedobacter sp. BAL39 (EDM38577; 66.6% identity) and a characterized α-galactosidase from Carnobacterium piscicola BA (AAL27305; 30.1%). Phylogenetic analysis revealed that GalA17 was similar to GH 36 α-galactosidases from symbiotic bacteria sharing two putative catalytic motifs, KWD and SDXXDXXXR, in which D480, S548, D549, and R556 were essential for α-galactosidase activity based on site-directed mutagenesis. Purified recombinant GalA17 showed apparent optimal activity at pH 5.5 and 45°C; exhibited strong resistance to digestion by trypsin, α-chymotrypsin, collagenase, and proteinase K; and efficiently hydrolyzed several synthetic and natural substrates (p-nitrophenyl-α-d-galactopyranoside, stachyose, melibiose, raffinose, soybean meal, locust bean gum, and guar gum).     

Cloning, expression, and purification of a recombinant cold-adapted beta-galactosidase from antarctic bacterium Pseudoalteromonas sp. 22b

The gram-negative antarctic bacterium Pseudoalteromonas sp. 22b, isolated from the alimentary tract of krill Thyssanoessa macrura, synthesizes an intracellular cold-adapted beta-galactosidase. The gene encoding this beta-galactosidase has been PCR amplified, cloned, expressed in Escherichia coli, purified, and characterized. The enzyme is active as a homotetrameric protein, and each monomer consists of 1028 amino acid residues. The enzyme was purified to homogeneity (50% recovery of activity) by using the fast, two-step procedure, including affinity chromatography on PABTG-Sepharose. Enzymatic properties of the recombinant protein are identical to those of native Pseudoalteromonas sp. 22b beta-galactosidase. The enzyme is cold-adapted and at 10 degrees C retains 20% of maximum activity. The purified enzyme displayed maximum activity close to 40 degrees C and at pH of 6.0-8.0. PNPG was its preferred substrate (58% higher activity than against ONPG). The enzyme was particularly thermolabile, losing all activities within 10 min at 50 degrees C. The hydrolysis of lactose in a milk assay revealed that 90% of milk lactose was hydrolyzed during 6 h at 30 degrees C and during 28 h at 15 degrees C. Because of its attributes, the recombinant Pseudoalteromonas sp. 22b beta-galactosidase could be applied at refrigeration temperatures for production of lactose-reduced dairy products.     

Purification and characterisation of a beta-galactosidase from Aspergillus aculeatus with activity towards (modified) exopolysaccharides from Lactococcus lactis subsp. cremoris B39 and B891

Beta-galactosidase from Aspergillus aculeatus was purified from a commercial source for its hydrolytic activity towards (modified) exopolysaccharides (EPSs) produced by Lactococcus lactis subsp. cremoris B39 and B891. The enzyme had a molecular mass of approximately 120 kDa, a pI between 5.3 and 5.7 and was optimally active at pH 5.4 and 55-60 degrees C. Based on the N-terminal amino acid sequence, the enzyme probably belongs to family 35 of the glycosyl hydrolases. The catalytic mechanism was shown to be retaining and transglycosylation products were demonstrated using lactose as a substrate. The beta-galactosidase was also characterised using its activity towards two EPSs having lactosyl side chains attached to different backbone structures. The enzyme degraded O-deacetylated EPS B891 faster than EPS B39. Furthermore, the presence of acetyl groups in EPS B891 slowed down the hydrolysing rate, but the enzyme was still able to release all terminally linked galactose.     

Production and properties of alpha-glucosidase from Lactobacillus acidophilus.

Lactobacillus acidophilus IFO 3532 was found to produce only intracellular alpha-glucosidase (alpha-D-glucoside glucohydrolase; EC 3.2.1.20). Maximum enzyme production was obtained in a medium containing 2% maltose as inducer at 37 degrees C and at an initial pH of 6.5. The enzyme was formed in the cytoplasm and accumulated as a large pool during the logarithmic growth phase. Enzyme production was strongly inhibited by 4 microM CuSO4, 40 microM CoCl2, and beef extract; MnSO4 and the presence of proteose peptone and yeast extract in the medium greatly enhanced enzyme production. A 16.6-fold purification of alpha-glucosidase was achieved by (NH4)2SO4 fractionation and DEAE-cellulose column chromatography. The enzyme showed high specificity for maltose. The Km for alpha-p-nitrophenyl-beta-D-glucopyranoside was 11.5 mM, and the Vmax for alpha-p-nitrophenyl-beta-D-glucopyranoside hydrolysis was 12.99 mumol/min per mg of protein. The optimal pH and temperature for enzyme activity were 5.0 and 37 degrees C, respectively. The enzyme activity was inhibited by Hg2+, Cu2+, Ni2+, Zn2+, Ca2+, Co2+, urea, rose bengal, and 2-iodoacetamide, whereas Mn2+, Mg2+, L-cysteine, L-histidine, Tris, and EDTA stimulated enzyme activity. Transglucosylase activity was present in the partially purified enzyme, and isomaltose was the only glucosyltransferase product. Amylase activity in the purified preparation was relatively weak, and no isomaltase activity was detected.     

Effect of low and high temperatures on chymotrypsin from Atlantic cod (Gadus morhua L.); comparison with bovine alpha-chymotrypsin

1. Cod chymotrypsin displays higher enzyme activity compared to bovine alpha-chymotrypsin when assayed at low temperatures (3-15 degrees C). 2. Both enzymes are inactivated when incubated at temperatures between 60 and 70 degrees C. 3. When incubated at 99 degrees C the cod enzyme retains about 50% of the initial activity measured at room temperature. 4. Preincubation at boiling temperature renders the cod chymotrypsin active at 70 degrees C whereas the bovine enzyme is rapidly inactivated.     

Purification and characterization of carboxymethyl cellulase from Bacillus sp. isolated from a paddy field

A microorganism hydrolyzing carboxymethyl cellulose was isolated from a paddy field and identified as Bacillus sp. Production of cellulase by this bacterium was found to be optimal at pH 6.5, 37 degrees C and 150 rpm of shaking. This cellulase was purified to homogeneity by the combination of ammonium sulphate precipitation, DEAE cellulose, and sephadex G-75 gel filtration chromatography. The cellulase was purified up to 14.5 fold and had a specific activity of 246 U/mg protein. The enzyme was a monomeric cellulase with a relative molecular mass of 58 kDa, as determined by SDS-PAGE. The enzyme exhibited its optimal activity at 50 degrees C and pH 6.0. The enzyme was stable in the pH range of 5.0 to 7.0 and its stability was maintained for 30 min at 50 degrees C and its activity got inhibited by Hg2+, Cu2+, Zn2+, Mg2+, Na2+, and Ca2+.     

In vitro biosynthesis of homogalacturonan by a membrane-bound galacturonosyltransferase from epicotyls of azuki bean

A membrane preparation of 7-d-old seedlings from azuki bean (Vigna angularis) contained galacturonosyltransferase (GalAT) capable of transferring galacturonic acid (GalA) from UDP-GalA into polygalacturonic acid (PGA) as an exogenous acceptor. The enzyme was maximally active at pH 6.8-7.8 and 25-35 degrees C in the presence of 5 mM Mn2+ and 0.5% (w/v) Triton X-100. Acid-soluble low-Mr (average Mr 10,000) PGA was a more efficient acceptor substrate than acid-insoluble polymer (Mr 70,000). The apparent Michaelis constants for UDP-GalA and low-Mr PGA were 0.14 mM and 0.02 mg/ml, respectively. Various pectins with different degrees of methyl-esterification (DE) were poor acceptors, and the enzyme activity tended to decrease with decreasing DE of the pectins. The transfer products from incubation of the enzyme with UDP-14C-GalA and the low-Mr PGA yielded 14C-GalA2 as the major product upon digestion with an endopolygalacturonase (EPGase), confirming the incorporation of GalA into PGA through contiguous alpha-1,4-linkages.     

Purification and characterization of a thermotolerant beta-galactosidase from Thermomyces lanuginosus.

A new inducible intracellular beta-galactosidase (EC 3.2.1.23) of the thermophilic fungus Thermomyces lanuginosus was purified by fractional salt precipitation, hydrophobic interaction, and anion exchange chromatography. The first 22 amino acid residues were determined by N-terminal sequencing. Electrophoretic investigations revealed a dimeric enzyme with a molecular mass of 75 to 80 kDa per identical subunit and an isoelectric point of 4.4 to 4.5. The native beta-galactosidase was identified as a glycoprotein by the enzyme-linked immunosorbent assay technique. The beta-galactosidase activity was optimal at pH 6.7 to 7.2, and the enzyme displayed stability between pH 6 and 9. It was completely stable at pH 6.8 and 47 degrees C for 2 h. After 191 h at 50 degrees C, the remaining beta-galactosidase activity of an enzyme fraction after salt precipitation was 58%. The beta-galactosidase hydrolyzed p- and o-NO2-phenyl-beta-D-galactopyranoside, lactose, lactulose, MeOH-beta-D-galactopyranoside, phenyl-beta-D-galactopyranoside, and p-NO2-phenyl-alpha-L-arabinopyranoside. The kinetic constants (Km) measured for p- and o-NO2-phenyl-beta-D-galactopyranoside and beta-lactose were 4.8, 11.3, and 18.2 mM, respectively.     

Purification and thermal characterization of a novel peroxidase from a local chick pea cultivar

A novel peroxidase isolated from a local chick pea (Cicer arietinum L.) cultivar (Balksar 2000) was purified by means of ammonium sulfate precipitation, DEAE-cellulose chromatography and two runs on gel filtration. The purified enzyme has a specific activity of 2045 U/mg with 17 % activity recovery. The molecular mass of the enzyme was estimated to be 39 kDa by SDS-polyacrylamide gel electrophoresis. Optimum pH and temperature of the enzyme were 5.5 and 45 degrees C respectively. The thermal denaturation of local chick pea peroxidase was studied in aqueous solution at temperatures ranging from 45 degrees C to 65 degrees C. The temperature of 50% inactivation of the enzyme was found to be 68 degrees C. The enthalpy (DeltaH*) and free energy (DeltaG*) of thermal denaturation of chick pea peroxidase were 101.4 and 103.4 k J/mol respectively at 65 degrees C.Metals like Zn2+, Mn2+, Hg2+, Co2+ and Al3+ slightly inhibited the peroxidase activity while Ca2+, Mg2+ and Ba2+ have no effect on enzyme activity. The high specific activity and thermal stability make chick pea peroxidase an alternative to horseradish peroxidase (HRP) in various applications.     

Characterization of a thermostable extracellular beta-galactosidase from a thermophilic fungus Rhizomucor sp

An extracellular beta-galactosidase from a thermophilic fungus Rhizomucor sp. has been purified to homogeneity by successive DEAE cellulose chromatography followed by gel filtration on Sephacryl S-300. The native molecular mass of the enzyme is 250,000 and it is composed of two identical subunits with molecular mass of 120,000. It is an acidic protein with a pI of 4.2. Purified beta-galactosidase is a glycoprotein and contains 8% neutral sugar. The optimum pH and temperature for enzyme activity are 4.5 and 60 degrees C, respectively. The enzyme is stable at 60 degrees C for 4 h, and has a t(1/2) of 150 min(-1) at 70 degrees C which is one of the highest reported for fungal beta-galactosidases. Substrate specificity studies indicated that the enzyme is specific for beta-linked galactose residues with a preference for p-nitrophenyl-beta-D-galactopyranoside (pNPG). The Km and Vmax values for the synthetic substrates pNPG and o-nitrophenyl-beta-D-galactopyranoside (oNPG) were 0.66 mM and 1.32 mM; and 22.4 mmol min(-1) mg(-1) and 4.45 mmol min(-1) mg(-1), respectively, while that for the natural substrate, lactose, was 50.0 mM and 12 mmol min(-1) mg(-1). The end product galactose and the substrate analogue isopropyl thiogalactopyranoside (ITPG) inhibited the enzyme with Ki of 2.6 mM and 12.0 mM, respectively. The energy of activation for the enzyme using pNPG and oNPG were 27.04 kCal and 9.04 kCal, respectively. The active site characterization studies using group-specific reagents revealed that a tryptophan and lysine residue play an important role in the catalytic activity of the enzyme.     

Adenosine diphosphate ribose transferase from baby-hamster kidney cells (BHK-21/C13). Characterization of the reaction and product.

Some properties of ADP-ribose transferase, and its reaction product, from BHK-21/C13 cells are described. Enzyme activity was found almost exclusively in nuclei (90%), with the remaining 10% located in the cytosolic fraction. The nuclear enzyme is chromatin-bound and requires bivalent cations, preferably Mg2+, a pH of 8.0 and a temperature of 25 degrees C for optimal activity. Chromatin preparations incorporated radioactivity from [14C]NAD+ into acid-insoluble material for about 60 min. Kinetics for substrate NAD+ utilization were not of Michaelis--Menten type; biphasic kinetics were shown from a double-reciprocal plot (1/reaction velocity against 1/[NAD+]) and from a 'Hofstee' plot (reaction velocity/[NAD+] against reaction velocity). The transferase is unstable in the absence of Mg2+ ions. It is inhibited by thymidine, nicotinamide and nicotinamide analogues, but not by ATP, which stimulates it at concentrations of 5 mM and above. The enzyme requires thiol groups for activity; it is readily inhibited by N-ethylmaleimide at 0.5 mM. The product of the reaction is stable under acid conditions at temperatures up to 25 degrees C, but it is hydrolysed by HClO4 at 70 degrees C. It is resistant to NaOH, but is cleaved from its attachment to protein with alkali into trichloroacetic acid-insoluble and -soluble components. On the basis of Cs2SO4- density-gradient analysis under denaturing conditions (gradients included urea and guanidinium hydrochloride), and analysis of the reaction product directly on hydroxyapatite, we conclude that most of the radioactive ADP-ribose residues are firmly bound to protein, presumably in covalent linkage. Hydroxyapatite-chromatographic analysis of ADP-ribose residues released from protein by alkaline digestion showed a spectrum of molecular sizes including mono-, oligo- and poly-(ADP-ribose), when chromatin was incubated initially with [14C]NAD+ for 10 min and then for a further 30 min after addition of excess non-radioactive NAD+, only about 10% of the radioactive mono-(ADP-ribose) could be 'chased' into longer-chain molecules. Hydroxyapatite analysis was also used to show that, whereas all ADP-ribose residues were released from protein with NaOH, only 50% of them were susceptible to hydroxylamine. These hydroxylamine-sensitive residues included all size classes, although mono-(ADP-ribose) predominated. Finally, there was an approximately equal distribution of ADP-ribose incorporated into HCl-soluble proteins (including the histones) and HCl-insoluble proteins (including the non-histone proteins) when chromatin was incubated with NAD+ up to 0.5 mM, but at higher NAD+ concentrations more ADP-ribose was incorporated into the HCl-soluble fraction (82% at 4.0 mM-NAD+).     

Induction of bphA, encoding biphenyl dioxygenase, in two polychlorinated biphenyl-degrading bacteria, psychrotolerant Pseudomonas strain Cam-1 and mesophilic Burkholderia strain LB400

We investigated induction of biphenyl dioxygenase in the psychrotolerant polychlorinated biphenyl (PCB) degrader Pseudomonas strain Cam-1 and in the mesophilic PCB degrader Burkholderia strain LB400. Using a counterselectable gene replacement vector, we inserted a lacZ-Gm(r) fusion cassette between chromosomal genes encoding the large subunit (bphA) and small subunit (bphE) of biphenyl dioxygenase in Cam-1 and LB400, generating Cam-10 and LB400-1, respectively. Potential inducers of bphA were added to cell suspensions of Cam-10 and LB400-1 incubated at 30 degrees C, and then beta-galactosidase activity was measured. Biphenyl induced beta-galactosidase activity in Cam-10 to a level approximately six times greater than the basal level in cells incubated with pyruvate. In contrast, the beta-galactosidase activities in LB400-1 incubated with biphenyl and in LB400-1 incubated with pyruvate were indistinguishable. At a concentration of 1 mM, most of the 40 potential inducers tested were inhibitory to induction by biphenyl of beta-galactosidase activity in Cam-10. The exceptions were naphthalene, salicylate, 2-chlorobiphenyl, and 4-chlorobiphenyl, which induced beta-galactosidase activity in Cam-10, although at levels that were no more than 30% of the levels induced by biphenyl. After incubation for 24 h at 7 degrees C, biphenyl induced beta-galactosidase activity in Cam-10 to a level approximately four times greater than the basal level in cells incubated with pyruvate. The constitutive level of beta-galactosidase activity in LB400-1 grown at 15 degrees C was approximately five times less than the level in LB400-1 grown at 30 degrees C. Thus, there are substantial differences in the effects of physical and chemical environmental conditions on genetic regulation of PCB degradation in different bacteria.