scholarly journals Antimicrobial Interventions to Reduce Shiga Toxin-Producing Escherichia coli (STEC) Surrogate Populations on Beef Striploins Intended for Blade Tenderization

2019 ◽  
Vol 3 (2) ◽  
Author(s):  
C. L. Thomas ◽  
H. Thippareddi ◽  
M. Rigdon ◽  
S. Kumar ◽  
R. W. McKee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl ◽  
Antimicrobial Treatment ◽  
Sodium Chlorite ◽  
Beef Industry ◽  
Anterior Portion ◽  
E Coli ◽  
Group Data ◽  
Blade Tenderization ◽  
Antimicrobial Interventions

ObjectivesBlade tenderization (BT) is used in the beef industry to improve tenderness of steaks prepared from subprimals but can translocate surface pathogens to the interior of meat. Application of antimicrobial solutions on the surface of subprimals prior to blade tenderization can reduce the risk of translocation of surface microorganisms. The objectives of this research were: 1) evaluate the efficacy of antimicrobial interventions applied to inoculated (surrogate Escherichia coli) beef striploins prior to blade tenderization; and 2) examine the transfer of E. coli from inoculated striploins to subsequent non-inoculated subprimals.Materials and MethodsThe anterior portion of whole muscle beef striploins (30.48 cm) were inoculated (lean side) across a 10 cm band with an approximately 8.00 log CFU/mL cocktail containing non-pathogenic, rifampicin-resistant surrogate STEC strains (BAA-1427, BAA-1428, BAA-1429, BAA-1430, and BAA-1431). The inoculated striploins were sprayed with (1) levulinic acid (5.0%) + sodium dodecyl sulfate (0.50%) (LVA+SDS), (2) peroxyacetic acid (2000 ppm; PAA; FCN 1666), (3) acidified sodium chlorite (1200 ppm; ASC), or (4) lactic acid (4.5%; LA) by passing through a spray cabinet and blade tenderized, along with an inoculated, non-sprayed control (CON). To evaluate the potential for cross-contamination of subsequent subprimals, an inoculated striploin (for each treatment) was blade tenderized followed by a non-inoculated beef striploin. For each striploin, surface and subsurface samples (2.54 cm wide) were collected from three different locations including the anterior, middle, and posterior end of each striploin. A total of 30 striploins across three replications were randomly assigned to treatment stratification. Sponge samples were also collected from the blade tenderizer (plate of the blade unit and blades) after each treatment group. Data were analyzed using Proc Mixed (SAS Inst., v.9.4; Cary, NC) as a completely randomized split-plot design. Microbial counts for all samples were log transformed and then analyzed for the main effects of antimicrobial treatment, location (anterior to posterior and surface or interior), and their interaction. Differences were considered significant at α ≤ 0.05.ResultsPAA was more effective in reducing E. coli populations (1.80 log CFU/g; P ≤ 0.05) and had lowest recovery of the microorganism from the striploin subsurface compared to other treatments, followed by LVA+SDS (1.00 log CFU/g). E. coli populations gradually decreased (P ≤ 0.05) on the surface and subsurface as sampling moved anterior to posterior. However, E. coli populations were similar (P > 0.05) on the posterior end of inoculated striploins and the anterior end of the subsequent, non-inoculated striploins, indicating transfer of microorganisms from one striploin to the following striploin. E. coli populations of 3.03 log CFU/cm2 and 2.47 log CFU/cm2 were recovered from the plate of the blade unit and the blades of the blade tenderizer. E. coli populations recovered from the plastic plate (3.46 log CFU/cm2) and blades (2.87 log CFU/cm2) of the blade tenderizer were the similar (P > 0.05) for all treatment groups except for PAA (1.41 log CFU/cm2 and 0.97 log CFU/cm2, respectively).ConclusionThese results showed that PAA and LVA+SDS can be used to improve the safety of blade tenderized beef.

Validation of Antimicrobial Interventions for Reducing Shiga Toxin–Producing Escherichia coli Surrogate Populations during Goat Slaughter and Carcass Chilling

2019 ◽  
Vol 82 (3) ◽  
pp. 364-370
Author(s):  
CHEVISE L. THOMAS ◽  
ALEXANDER M. STELZLENI ◽  
ANGELA G. RINCON ◽  
SANJAY KUMAR ◽  
MACC RIGDON ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Sodium Dodecyl ◽  
Small Ruminants ◽  
The United States ◽  
Antimicrobial Treatment ◽  
Goat Meat ◽  
E Coli ◽  
Ruminant Species ◽  
Antimicrobial Interventions

ABSTRACT Demand and consumption of goat meat is increasing in the United States due to an increase in ethnic populations that prefer goat meat. As ruminant animals, goats are known reservoirs for Shiga toxin–producing Escherichia coli (STEC) and proper handling, especially during slaughter, is imperative to reduce the likelihood of carcass and meat contamination. However, the majority of antimicrobial intervention studies during the slaughter of ruminant species have focused on beef, highlighting the need for validation studies targeting small ruminants, such as goats, during slaughter and chilling procedures. The objective of this research was to evaluate 4.5% lactic acid (LA; pH 2.1), peroxyacetic acid (PAA; 400 ppm; pH 4.7), a hydrochloric and citric acid blend (Citrilow [CL]; pH 1.2), 5% levulinic acid plus 0.5% sodium dodecyl sulfate (LVA+SDS; pH 2.60), and a nontreated control (CON) for their efficacy in reducing STEC surrogates and their effect on carcass color from slaughter through 24-h chill. Fifteen goat carcasses across three replications were inoculated with a five-strain cocktail (ca. 5 log CFU/cm2 attachment), containing rifampin-resistant surrogate E. coli (BAA-1427, BAA-1428, BAA-1429, BAA-1430, and BAA-1431) and were randomly assigned to an antimicrobial treatment. Antimicrobials were applied prechill and 24 h postchill. Mean log reductions achieved after prechill treatment with LA, PAA, CL, and LVA+SDS were 2.00, 1.86, 2.26, and 1.90 log CFU/cm2, respectively. Antimicrobial treatment after the 24-h chilling, resulted in additional reductions of surrogate E. coli by 0.99, 1.03, 1.94, and 0.47 log CFU/cm2 for LA, PAA, CL, and LVA+SDS, respectively. Antimicrobial treatments did not impact goat carcass objective color (L* and a*), except for b*. The antimicrobials tested in this study were able to effectively reduce surrogate STEC populations during slaughter and subsequent chilling without compromising carcass color.

Decontamination of Beef Subprimal Cuts Intended for Blade Tenderization or Moisture Enhancement

2007 ◽  
Vol 70 (5) ◽  
pp. 1174-1180 ◽  
Author(s):  
C. E. HELLER ◽  
J. A. SCANGA ◽  
J. N. SOFOS ◽  
K. E. BELK ◽  
W. WARREN-SERNA ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lactic Acid ◽  
Hot Water ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Low Concentrations ◽  
Blade Tenderization ◽  
Antimicrobial Interventions

The prevalence of Escherichia coli O157:H7 on beef subprimal cuts intended for mechanical tenderization was evaluated. This evaluation was followed by the assessment of five antimicrobial interventions at minimizing the risk of transferring E. coli O157:H7 to the interior of inoculated subprimal cuts during blade tenderization (BT) or moisture enhancement (ME). Prevalence of E. coli O157:H7 on 1,014 uninoculated beef subprimals collected from six packing facilities was 0.2%. Outside round pieces inoculated with E. coli O157:H7 at 104 CFU/100 cm2 were treated with (i) no intervention, (ii) surface trimming, (iii) hot water (82°C), (iv) warm 2.5% lactic acid (55°C), (v) warm 5.0% lactic acid (55°C), or (vi) 2% activated lactoferrin followed by warm 5.0% lactic acid (55°C) and then submitted to BT or ME. Prevalence (n = 196) of internalized (BT and ME) E. coli O157:H7 was 99%. Enumeration of E. coli O157:H7 (n = 192) revealed mean surface reductions of 0.93 to 1.10 log CFU/100 cm2 for all antimicrobial interventions. E. coli O157:H7 was detected on 3 of the 76 internal BT samples and 73 of the 76 internal ME samples. Internal ME samples with no intervention had significantly higher mean E. coli O157:H7 populations than did those internal samples treated with an intervention, but there were no significant differences in E. coli O157:H7 populations among internal BT samples. Results of this study demonstrate that the incidence of E. coli O157:H7 on the surface of beef subprimal cuts is low and that interventions applied before mechanical tenderization can effectively reduce the transfer of low concentrations of E. coli O157:H7 to the interior of beef subprimal cuts.

Efficacy of Chemical Interventions against Escherichia coli O157:H7 and Multidrug-Resistant and Antibiotic-Susceptible Salmonella on Inoculated Beef Trimmings

2012 ◽  
Vol 75 (11) ◽  
pp. 1960-1967 ◽  
Author(s):  
IFIGENIA GEORNARAS ◽  
HUA YANG ◽  
GALATIOS MOSCHONAS ◽  
MATTHEW C. NUNNELLY ◽  
KEITH E. BELK ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Salmonella Typhimurium ◽  
Chemical Treatment ◽  
Sodium Metasilicate ◽  
Multidrug Resistant ◽  
Antimicrobial Treatment ◽  
Sodium Chlorite ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Salmonella Newport

Studies were conducted to compare the decontamination efficacy of six chemical treatments against Escherichia coli O157:H7 and multidrug-resistant and antibiotic-susceptible Salmonella inoculated on beef trimmings. The inocula, comprising four-strain mixtures of rifampin-resistant E. coli O157:H7 and antibiotic-susceptible or multidrug-resistant (MDR and/or MDR-AmpC) Salmonella Newport and Salmonella Typhimurium, were inoculated (3 log CFU/cm2) separately onto samples (10 by 5 by 1 cm) derived from beef chuck rolls. Samples were left untreated (control), were immersed for 30 s in acidified sodium chlorite (0.1%, pH 2.5), peroxyacetic acid (0.02%, pH 3.8), sodium metasilicate (4%, pH 12.6), Bromitize Plus (0.0225% active bromine, pH 6.6), or AFTEC 3000 (pH 1.2), or were immersed for 5 s in SYNTRx 3300 (pH 1.0). Levels of surviving Salmonella on treated trimmings were not influenced by serotype or antibiotic resistance phenotype and were generally similar (P > 0.05) or lower (P < 0.05) than levels of surviving E. coli O157:H7 regardless of antimicrobial treatment. Overall, depending on chemical treatment (reductions within each chemical treatment were similar among all tested inocula), initial counts of E. coli O157:H7 (2.7 to 3.1 log CFU/cm2) were reduced (P < 0.05) by 0.2 to 1.4 log CFU/cm2. Similarly, initial counts of the tested Salmonella inocula (2.8 to 3.3 log CFU/cm2) were reduced (P < 0.05) by 0.4 to 1.4 (Salmonella Newport, antibiotic susceptible), 0.3 to 1.4 (Salmonella Newport, MDRAmpC), 0.2 to 1.5 (Salmonella Typhimurium, antibiotic susceptible), 0.4 to 1.3 (Salmonella Typhimurium, MDR), and 0.4 to 1.5 (Salmonella Typhimurium, MDR-AmpC) log CFU/cm2, depending on antimicrobial treatment. Reductions obtained with sodium metasilicate were 1.3 to 1.5 log CFU/cm2, regardless of inoculum, and reductions obtained with the five remaining antimicrobial treatments were 0.2 to 0.7 log CFU/cm2 (depending on treatment). Findings of this study should be useful to regulatory authorities and the meat industry as they consider Salmonella contamination on beef trimmings.

scholarly journals Evaluation of Commonly Used Antimicrobial Interventions for Fresh Beef Inoculated with Shiga Toxin–Producing Escherichia coli Serotypes O26, O45, O103, O111, O121, O145, and O157:H7†

2012 ◽  
Vol 75 (7) ◽  
pp. 1207-1212 ◽  
Author(s):  
NORASAK KALCHAYANAND ◽  
TERRANCE M. ARTHUR ◽  
JOSEPH M. BOSILEVAC ◽  
JOHN W. SCHMIDT ◽  
RONG WANG ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lactic Acid ◽  
Shiga Toxin ◽  
Hot Water ◽  
Sodium Chlorite ◽  
Meat Industry ◽  
Peroxyacetic Acid ◽  
E Coli ◽  
Low Level ◽  
Antimicrobial Interventions

Although numerous antimicrobial interventions targeting Escherichia coli O157:H7 have been developed and implemented to decontaminate meat and meat products during the harvesting process, the information on efficacy of these interventions against the so-called Big Six non-O157 Shiga toxin–producing E. coli (STEC) strains is limited. Prerigor beef flanks (160) were inoculated to determine if antimicrobial interventions currently used by the meat industry have a similar effect in reducing non-O157 STEC serogroups O26, O45, O103, O111, O121, and O145 compared with E. coli O157:H7. A high (104 CFU/cm2) or a low (101 CFU/cm2) inoculation of two cocktail mixtures was applied to surfaces of fresh beef. Cocktail mixture 1 was composed of O26, O103, O111, O145, and O157, while cocktail mixture 2 was composed of O45, O121, and O157. The inoculated fresh beef flanks were subjected to spray treatments by the following four antimicrobial compounds: acidified sodium chlorite, peroxyacetic acid, lactic acid, and hot water. High-level inoculation samples were enumerated for the remaining bacteria populations after each treatment and compared with the untreated controls, while low-level inoculation samples were chilled for 48 h at 4°C before enrichment, immunomagnetic separation, and isolation. Spray treatments with hot water were the most effective, resulting in mean pathogen reductions of 3.2 to 4.2 log CFU/cm2, followed by lactic acid. Hot water and lactic acid also were the most effective interventions with the low-level inoculation on surfaces of fresh beef flanks after chilling. Peroxyacetic acid had an intermediate effect, while acidified sodium chlorite was the least effective in reducing STEC levels immediately after treatment. Results indicate that the reduction of non-O157 STEC by antimicrobial interventions on fresh beef surfaces were at least as great as for E. coli O157:H7. However, the recovery of these low inoculation levels of pathogens indicated that there is no single intervention to eliminate them.

Structure of the O-chain polysaccharide of the phenol-phase soluble lipopolysaccharide of Escherichia coli 0:157:H7

1986 ◽  
Vol 64 (1) ◽  
pp. 21-28 ◽  
Author(s):  
Malcolm B. Perry ◽  
Leann MacLean ◽  
Douglas W. Griffith
Keyword(s):  
Escherichia Coli ◽  
Brucella Abortus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extraction Procedure ◽  
Periodate Oxidation ◽  
Cross Reactivity ◽  
E Coli ◽  
Structure Formula ◽  
Phenol Water

The phenol-phase soluble lipopolysaccharide isolated from Escherichia coli 0:157 by the hot phenol–water extraction procedure was shown by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, periodate oxidation, methylation, and 13C and 1H nuclear magnetic resonance studies to be an unbranched linear polysaccharide with a tetrasaccharide repeating unit having the structure:[Formula: see text]The serological cross-reactivity of E. coli 0:157 with Brucella abortus, Yersinia enterocolitica (serotype 0:9), group N Salmonella, and some other E. coli species can be related immunochemically to the presence of 1,2-glycosylated N-acylated 4-amino-4,6-dideoxy-α-D-mannopyranosyl residues in the O-chains of their respective lipopolysaccharides.

Two Novel Mutations Associated with Polymyxin-B Resistance in a Pandemic Lineage of Uropathogenic Escherichia coli of the Sequence Type 69

Chemotherapy ◽  
2021 ◽  
pp. 1-7
Author(s):  
Carla Adriana dos Santos ◽  
Rodrigo Tavanelli Hernandes ◽  
Marcos Paulo Vieira Cunha ◽  
Filipe Onishi Nagamori ◽  
Claudia Regina Gonçalves ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Susceptibility Testing ◽  
Polymyxin B ◽  
Antimicrobial Treatment ◽  
Sequence Type ◽  
Broth Microdilution ◽  
Economic Costs ◽  
Novel Mutations ◽  
E Coli ◽  
Total Inhibition

<b><i>Background:</i></b> Uropathogenic <i>Escherichia coli</i> (UPEC) are frequent pathogens worldwide, impacting on the morbidity and economic costs associated with antimicrobial treatment. <b><i>Objectives:</i></b> We report two novel mutations associated with polymyxin-B resistance in an UPEC isolate collected in 2019. <b><i>Methods:</i></b> Isolate was submitted to antimicrobial susceptibility testing including broth microdilution for polymyxin B. Whole genome was sequenced and analyzed. <b><i>Results:</i></b> Polymyxin-B total inhibition occurred at 16 mg/L (resistant). UPEC isolate was assigned to the phylogroup D, serotype O117:H4, and Sequence Type 69. <i>mcr</i> genes were not detected, but two novel mutations in the <i>pmrA/basS</i> (A80S) and <i>pmrB/</i>basR (D149N) genes were identified. <b><i>Conclusions:</i></b> The occurrence of non-<i>mcr</i> polymyxin resistance in <i>E. coli</i> from extraintestinal infections underscores the need of a continuous surveillance of this evolving pathogen.

scholarly journals Acetate and Formate Stress: Opposite Responses in the Proteome of Escherichia coli

2001 ◽  
Vol 183 (21) ◽  
pp. 6466-6477 ◽  
Author(s):  
Christopher Kirkpatrick ◽  
Lisa M. Maurer ◽  
Nikki E. Oyelakin ◽  
Yuliya N. Yoncheva ◽  
Russell Maurer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Opposite Effect ◽  
Stress Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Acid Resistance ◽  
Luria Broth ◽  
Acetyl Coa ◽  
Fermentation Products ◽  
E Coli

ABSTRACT Acetate and formate are major fermentation products ofEscherichia coli. Below pH 7, the balance shifts to lactate; an oversupply of acetate or formate retards growth. E. coli W3110 was grown with aeration in potassium-modified Luria broth buffered at pH 6.7 in the presence or absence of added acetate or formate, and the protein profiles were compared by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Acetate increased the steady-state expression levels of 37 proteins, including periplasmic transporters for amino acids and peptides (ArtI, FliY, OppA, and ProX), metabolic enzymes (YfiD and GatY), the RpoS growth phase regulon, and the autoinducer synthesis protein LuxS. Acetate repressed 17 proteins, among them phosphotransferase (Pta). An ackA-pta deletion, which nearly eliminates interconversion between acetate and acetyl-coenzyme A (acetyl-CoA), led to elevated basal levels of 16 of the acetate-inducible proteins, including the RpoS regulon. Consistent with RpoS activation, the ackA-pta strain also showed constitutive extreme-acid resistance. Formate, however, repressed 10 of the acetate-inducible proteins, including the RpoS regulon. Ten of the proteins with elevated basal levels in the ackA-ptastrain were repressed by growth of the mutant with formate; thus, the formate response took precedence over the loss of theackA-pta pathway. The similar effects of exogenous acetate and the ackA-pta deletion, and the opposite effect of formate, could have several causes; one possibility is that the excess buildup of acetyl-CoA upregulates stress proteins but excess formate depletes acetyl-CoA and downregulates these proteins.

scholarly journals The Effect Ultrasound and Surfactants on Nanobubbles Efficacy against Listeria innocua and Escherichia coli O157:H7, in Cell Suspension and on Fresh Produce Surfaces

Foods ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2154
Author(s):  
Shamil Rafeeq ◽  
Reza Ovissipour
Keyword(s):  
Escherichia Coli ◽  
Cell Suspension ◽  
Sodium Dodecyl ◽  
Listeria Innocua ◽  
Wash Water ◽  
Tween 20 ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Bacterial Removal ◽  
The Impact

Removing foodborne pathogens from food surfaces and inactivating them in wash water are critical steps for reducing the number of foodborne illnesses. In this study we evaluated the impact of surfactants on enhancing nanobubbles’ efficacy on Escherichia coli O157:H7, and Listeria innocua removal from spinach leaves. We evaluated the synergistic impact of nanobubbles and ultrasound on these two pathogens inactivation in the cell suspension. The results indicated that nanobubbles or ultrasound alone could not significantly reduce bacteria in cell suspension after 15 min. However, a combination of nanobubbles and ultrasonication caused more than 6 log cfu/mL reduction after 15 min, and 7 log cfu/mL reduction after 10 min of L. innocua and E. coli, respectively. Nanobubbles also enhanced bacterial removal from spinach surface in combination with ultrasonication. Nanobubbles with ultrasound removed more than 2 and 4 log cfu/cm2 of L. innocua and E. coli, respectively, while ultrasound alone caused 0.5 and 1 log cfu/cm2 of L. innocua and E. coli reduction, respectively. No reduction was observed in the solutions with PBS and nanobubbles. Adding food-grade surfactants (0.1% Sodium dodecyl sulfate-SDS, and 0.1% Tween 20), did not significantly enhance nanobubbles efficacy on bacterial removal from spinach surface.

scholarly journals Expression in Escherichia coli of the human fibrinogen B beta chain and its cleavage by thrombin

Blood ◽  
1989 ◽  
Vol 73 (5) ◽  
pp. 1202-1206 ◽  
Author(s):  
MG Bolyard ◽  
ST Lord
Keyword(s):  
Escherichia Coli ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Dependent Manner ◽  
Beta Chain ◽  
Gamma Chain ◽  
Human Fibrinogen ◽  
E Coli ◽  
Sds Page

Abstract The human fibrinogen B beta chain was expressed in Escherichia coli to study the functions of fibrinogen associated with this subunit. Recombinant B beta chains were expressed at 100 ng/mL in an IPTG- dependent manner. A first cistron sequence, inserted into the expression vector 5′ to the B beta chain cDNA, was required to express the protein. Recombinant B beta chains were expressed within five minutes after induction with IPTG and were soluble in physiologic buffers. The recombinant B beta chains migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at a rate identical to B beta chains from fibrinogen treated with N-glycanase. Recombinant B beta chains were cleaved by thrombin, as demonstrated by the loss of cross-reactivity with a monoclonal antibody (MoAb) specific for the undigested B beta 1–42 fragment. The levels of expression of the B beta chain were much lower than those reported previously for the gamma chain of fibrinogen expressed in a similar vector in E coli. However, these levels are sufficient to allow further characterization of this fibrinogen subunit.

scholarly journals Cloning, Nucleotide Sequence, and Overexpression of the l-Rhamnose Isomerase Gene from Pseudomonas stutzeri in Escherichia coli

2004 ◽  
Vol 70 (6) ◽  
pp. 3298-3304 ◽  
Author(s):  
Khim Leang ◽  
Goro Takada ◽  
Akihiro Ishimura ◽  
Masashi Okita ◽  
Ken Izumori
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Pseudomonas Stutzeri ◽  
Fold Increase ◽  
E Coli ◽  
Apparent Homogeneity ◽  
Volumetric Yield

ABSTRACT The gene encoding l-rhamnose isomerase (l-RhI) from Pseudomonas stutzeri was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the l-RhI gene revealed an open reading frame of 1,290 bp coding for a protein of 430 amino acid residues with a predicted molecular mass of 46,946 Da. A comparison of the deduced amino acid sequence with sequences in relevant databases indicated that no significant homology has previously been identified. An amino acid sequence alignment, however, suggested that the residues involved in the active site of l-RhI from E. coli are conserved in that from P. stutzeri. The l-RhI gene was then overexpressed in E. coli cells under the control of the T5 promoter. The recombinant clone, E. coli JM109, produced significant levels of l-RhI activity, with a specific activity of 140 U/mg and a volumetric yield of 20,000 U of soluble enzyme per liter of medium. This reflected a 20-fold increase in the volumetric yield compared to the value for the intrinsic yield. The recombinant l-RhI protein was purified to apparent homogeneity on the basis of three-step chromatography. The purified recombinant enzyme showed a single band with an estimated molecular weight of 42,000 in a sodium dodecyl sulfate-polyacrylamide gel. The overall enzymatic properties of the purified recombinant l-RhI protein were the same as those of the authentic one, as the optimal activity was measured at 60�C within a broad pH range from 5.0 to 11.0, with an optimum at pH 9.0.

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