Heterologous expression and stability of the Escherichia coli β-galactosidase gene in Streptococcus lactis by translation fusion

An Escherichia coli/Streptococcus lactis shuttle vector for creating translation fusions to the E. coli galactosidase (lacZ) has been constructed. Random S. lactis chromosomal DNA fragments inserted upstream of the lacZ gene and in frame promote the expression of active galactosidase in both E. coli and S. lactis. One fusion pSK216 has been characterized by Western blotting to reveal a fusion protein of 116 kd. S. lactis LM0230, a plasmidless lac⁻ derivative of S. lactis C2 when carrying pSK216, exhibits a Lac⁺ phenotype suggesting the presence of a chromosomally encoded lactose permease. Further evidence for this lactose permease system is provided by assay of lactose uptake by S. lactis LM0230. In the absence of selection pSK216 was less stable in S. lactis LM0230 than its derivative pSK41. The growth rate of S. lactis LM0230 carrying pSK216 is slower in comparison to the plasmid-free strain. Two genetic events were observed, the deletion of the lacZ gene and the loss of the entire plasmid. These results indicate that recombinant plasmids are unstable in S. lactis and imply that there is an obstacle in the genetic engineering of lactic acid bacteria.     

Purification and characterization of β-agarase from Paenibacillus sp.

β-Agarase produced by Paenibacillus sp. WL (agarase WL) was purified using a combination of ammonium sulfate precipitation, DEAE-ion exchange, and gel-filtration chromatography. The purity of the agarase was increased by 11.9× with a recovery of 5.1% and a specific activity of 4,670.1 U/mg of protein. The molecular mass of the purified agarase was approximately 30 kDa (SDS-PAGE). The agarase was stable at temperature below 50°C and the favorable agar-hydrolysis activity was at 40°C. The agarase was active in the range of pH 5.0 to 8.0, and the optimal agar-hydrolysis pH value was approximately 6.0. Metal ions normally found in seawater (Na⁺, K⁺, Ca²⁺, Mg²⁺, and Al³⁺) could activate agarase WL. The Michaelis-Menten constant K _m and maximal reaction velocity V _max of purified agarase WL were 3.22 mg/mL and 41.5 μg/mL·min, respectively. The agarase WL was highly agar specific.     

Antimicrobial susceptibility and characterization of extended-spectrum β-lactamases in Escherichia coli isolated from bovine mastitic milk in South Korea from 2012 to 2015

In this study, we aimed to assess trends in antimicrobial resistance and to investigate the characteristics of extended-spectrum β-lactamase (ESBL)-producing isolates from bovine mastitic milk from 2012 to 2015. A total of 374 Escherichia coli isolates were analyzed (154 in 2012, 113 in 2013, 76 in 2014, and 31 in 2015). No consistent trends in antimicrobial resistance of E. coli isolates occurred during the 4-yr period. The most frequently observed resistance was tetracycline (23.3%), followed by streptomycin (17.1%), ampicillin (16.6%), neomycin (11.8%), and trimethoprim/sulfamethoxazole (11.2%). Multidrug resistance was observed in 15.5% of isolates. Among these isolates, 15 (4.0%) carried one or more bla_CTX-M and AmpC ESBL genes from 11 different farms, including bla_CTX-M-15 at 4 farms, bla_CTX-M-3 at 2 farms, bla_CTX-M-1 at 3 farms, and bla_CMY-2 at 3 farms. This study is the first report of bla_CTX-M-3-producing E. coli in dairy milk. Transfer of ESBL was observed in 3 bla_CTX-M-3-producing isolates, 1 bla_CTX-M-1-producing isolate, and all 3 bla_CMY-2-producing isolates. Almost all bla_CTX-M-15 and bla_CTX-M-1 genes possessed an insertion sequence, ISECP1, upstream of the bla_CTX-M gene. Identical pulsed-field gel electrophoresis profiles were also observed in bla_CTX-M-producing E. coli from the same farm. These results suggested that ESBL might spread by both clonal and horizontal spread in dairy farms in South Korea. Although no significant changes occurred in the antimicrobial resistance of E. coli during the 4-yr study period, the resistance rates and presence of ESBL were high compared with those in other countries. Thus, these findings suggest the importance of control measures for E. coli, particularly ESBL-producing bacteria, on dairy farms to reduce treatment failure and transmission to humans.     

Double knockout of β-lactamase and cephalosporin acetyl esterase genes from Escherichia coli reduces cephalosporin C decomposition

The phenomenon of CPC decomposition occurs in Escherichia coli JM105/pMKC-sCPCacy during the one-step enzymatic conversion of cephalosporin C (CPC) into 7-aminocephalosporanic acid (7-ACA) by CPC acylase (sCPCAcy) for synthesis of cephalosporin antibiotics. E. coli JM105/pMKC-sCPCacy can constitutively produce sCPCacy as a fusion protein with maltose binding protein (MBP). Control experiments verified that the cell lysis solution from the host E. coli JM105 resulted in CPC decomposition by approximately 15%. Two miscellaneous enzymes, β-lactamase (AmpC) and cephalosporin acetyl esterase (Aes), are believed to play a major role in the degradation of CPC. Using the Red recombination system, the genes ampC, aes or both ampC and aes were knocked out from the chromosome of E. coli JM105 to generate the engineers: E. coli JM105(ΔampC), E. coli JM105(Δaes) and E. coli JM105(ΔampC, Δaes). The CPC decomposition was reduced to 12.2% in E. coli JM105(Δaes), 1.3% in E. coli JM105(ΔampC), and even undetectable in ampC-aes double knockout cells of E. coli JM105(ΔampC, Δaes). When catalyzed by crude MBP-sCPCAcy isolated from E. coli JM105(ΔampC, Δaes)/pMKC-sCPCacy (3377U·l(-1)), the CPC utilization efficiency increased to 98.4% from the original 88.7%. Similar results were obtained for the ampC-aes double knockout host derived from E. coli JM109(DE3) and the CPC utilization efficiency enhanced to 99.3% in the catalysis of crude sCPCAcy harvested from E. coli JM109(DE3, ΔampC, Δaes)/pET28-sCPCacy.     

Use of β-cyclodextrin to decrease the level of cholesterol in milk fat

This study was carried out to determine optimum conditions (β-cyclodextrin concentration, mixing time, and holding time) for cholesterol removal from pasteurized nonhomogenized milk at 4°C on a commercial scale by adding β-cyclodextrin in a specially designed bulk mixer tank. The β-cyclodextrin (0.4, 0.6, 0.8, and 1.0%) removed from 65.42 to 95.31% of cholesterol at 4°C in 20 min. Treatment of milk with 0.8 and 1.0% (wt/vol) β-cyclodextrin was no better than treatment with 0.6% β-cyclodextrin. Maximum cholesterol removal was seen with 6 h of treatment. The β-cyclodextrin cholesterol complex was precipitated from milk during 20 min without stirring at 4°C and removed by centrifugation. After separating the milk, approximately 0.35% of residual β-cyclodextrin remained in the skim fraction and 0.1% in the cream from milk treated with 0.6% β-cyclodextrin. The rest of the β-cyclodextrin was complexed with the cholesterol and eliminated via the discharger of the separator. Individual fatty acid and triglyceride compositions did not differ between control milk and milk treated with 0.6% β-cyclodextrin.     

Prevalence of extended-spectrum and AmpC β-lactamase-producing Escherichia coli in young calves on Dutch dairy farms

In young calves on dairy farms the animal prevalence of extended-spectrum and AmpC β-lactamase-producing Escherichia coli (ESBL/AmpC-EC) is significantly higher compared with the animal prevalence in young stock and dairy cows. Hitherto it was unknown at what age antimicrobial resistant bacteria appear for the first time in the gut of calves on dairy farms, and how long these infections persist. The aim of this study was to examine the prevalence of ESBL/AmpC-EC, the number of excreted ESBL/AmpC-EC (in cfu/g of feces), as well as the ESBL/AmpC genotypes in young dairy calves (0–21 d of age) and the variation of these parameters between calves of different ages. Next to this, the course of shedding ESBL/AmpC-EC during the first year in dairy calves was studied. In a cross-sectional study, fecal samples from 748 calves, from 0 to 88 d of age, on 188 Dutch dairy farms were collected. The prevalence of calves testing positive for ESBL/AmpC-EC in a phenotypic assay was determined for different age categories (per 2 d of age). Positive samples were subjected to a semiquantitative test to determine the numbers of ESBL/AmpC-EC per gram of feces and for a selection of ESBL/AmpC-EC isolates the ESBL/AmpC genotype was determined. Ten of the 188 farms were selected for a longitudinal study based on the presence of at least 1 female calf with ESBL/Amp-EC in the cross-sectional study. These farms were additionally visited 3 times with a 4-mo interval. All calves that were sampled in the cross-sectional study were, if still present, resampled during the follow-up visits. Results show that from the day of birth ESBL/AmpC-EC can be present in the gut of calves. The phenotypic prevalence of ESBL/AmpC-EC was 33.3% in 0- to 21-d-old calves and 28.4% in 22- to 88-d-old calves. The prevalence of ESBL/AmpC-EC positive calves varied per age category among calves up to 21 d of age: significant increases and decreases at an early age were shown. Results of the longitudinal study show that after 4, 8, and 12 mo the prevalence of ESBL/AmpC-EC positive calves dropped to 3.8% (2/53), 5.8% (3/52), and 2.0% (1/49), respectively. This indicates that early gut colonization in young calves with ESBL/AmpC-EC is transient and does not lead to long-term shedding of these bacteria.     

Electrochemical nucleic acid-based sensors for detection of Escherichia coli and Shiga toxin-producing E. coli—Review of the recent developments

Escherichia coli are a group of bacteria that are a natural part of the intestinal flora of warm-blooded animals, including humans. Most E. coli are nonpathogenic and essential for the normal function of a healthy intestine. However, certain types, such as Shiga toxin-producing E. coli (STEC), which is a foodborne pathogen, can cause a life-threatening illness. The development of point-of-care devices for the rapid detection of E. coli is of significant interest with regard to ensuring food safety. The most suitable way to distinguish between generic E. coli and STEC is by using nucleic acid-based detection, focusing on the virulence factors. Electrochemical sensors based on nucleic acid recognition have attracted much attention in recent years for use in pathogenic bacteria detection. This review has summarized nucleic acid-based sensors for the detection of generic E. coli and STEC since 2015. First, the sequences of the genes used as recognition probes are discussed and compared to the most recent research regarding the specific detection of general E. coli and STEC. Subsequently, the collected literature regarding nucleic acid-based sensors is described and discussed. The traditional sensors were divided into four categories such as gold, indium tin oxide, carbon-based electrodes, and those using magnetic particles. Finally, we summarized the future trends in nucleic acid-based sensor development for E. coli and STEC including some examples of fully integrated devices.     

Use of propidium monoazide and quantitative PCR for differentiation of viable Escherichia coli from E. coli killed by mild or pasteurizing heat treatments

Suspensions of Escherichia coli in peptone water were heated at temperatures between 52 and 90 °C, inclusive. Samples withdrawn at suitable times were not or were treated with propidium monoazide (PMA) or deoxycholate then PMA before extraction of DNA. DNA was quantified by real-time PCR for estimation of the numbers of E. coli from which template DNA for the PCR was obtained. Numbers of viable E. coli in suspensions at the times of sampling were determined from plate counts. For samples from suspensions heated at temperatures ≥52 ≤ 72 °C, PCR cycle threshold (Ct) values were little or no different for DNA from corresponding samples that were or were not treated with PMA. PMA treatment of samples heated to ≥80 °C largely inactivated E. coli DNA for PCR. When samples heated to ≤72 °C were treated with deoxycholate before treatment with PMA, Ct values for treated samples were greater than the Ct values for the corresponding untreated samples. Similar results were obtained with E. coli suspended in milk or fluid from ground beef pummeled with diluent. The results indicate that cells killed by heating to ≥80 °C are permeable to PMA, but most cells killed by heating to ≤72 °C are not. However, treatment with deoxycholate renders a substantial fraction of the latter cells permeable to PMA. Numbers of viable or dead E. coli can then be estimated from Ct values for samples not treated or treated with deoxycholate and PMA, provided viable cells are ≥1% of the total.     

Extraction of β-carotene produced from yeast Rhodosporidium sp. and its heat stability

This work determined the characteristics of β-carotene produced from Rhodosporidium sp. isolated from citrus fruits, and an effective extraction method was established. To extract β-carotene from the isolated Rhodosporidium sp., the cell walls were destroyed using dimethyl sulfoxide (DMSO) solution with and without the use of glass beads and a sonicator. Extracted β-carotene was identified by high performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS) with a β-carotene standard. The yields of β-carotene extracted in DMSO, DMSO and glass beads, and DMSO with a sonicator were 3.371, 5.112, and 3.301 μg/mL, respectively. Isolated β-carotene was relatively heat stable, with 80% of the viable molecules remaining at 80°C.     

The role of olive β-glucosidase in shaping the phenolic profile of virgin olive oil

The main objective of this work was to investigate the biochemical factors directly affecting the phenolic profile of virgin olive oil (VOO) such as the content of phenolic glycosides and the β-glucosidase activity present in the olive fruit. The phenolic compositions of VOO from two olive cultivars, Arbequina and Picual, were studied throughout the ripening phase. Picual oils displayed significantly higher phenolic content than Arbequina oils at all ripening stages; however, the total phenolic content of Arbequina fruits was consistently higher than that of Picual fruits. The most abundant phenolic glucosides found in olive fruits were oleuropein, present in both cultivars, and demethyloleuropein, only detected in Arbequina fruits. Nevertheless, the content of phenolic glycosides can hardly be regarded as a limiting factor given that the total secoiridoid content in VOOs represents an average of 1–4% of the secoiridoid glycosides present in the fruits. Although the level of β-glucosidase activity does not seem to be a limiting factor in these two cultivars, experimental data on the selectivity of the enzyme towards olive phenolic glucosides and its product specificity suggest that olive β-glucosidase plays a critical role in shaping the phenolic profile of VOO. A hypothesis on the biochemical formation of the dialdehydic form of decarboxymethyloleuropein (3,4-DHPEA-EDA) from oleuropein is proposed.     

Inactivation of Staphylococcus aureus and Escherichia coli by the synergistic action of high hydrostatic pressure and dissolved CO₂

This study focused on the synergistic inactivation effects of combined treatment of HHP and dissolved CO₂ on microorganisms. The aim was to reduce the treatment pressure of the traditional HHP technology and make it more economically feasible. The combined treatment showed a strong bactericidal effect on Staphylococcus aureus and Escherichia coli in liquid culture, which usually have high levels of barotolerance under pressure alone. To identify the influence of CO₂, a new setup to dissolve, retain and measure the concentration of CO₂ was constructed. The results demonstrated that an inactivation rate of more than 8 log units was obtained for E. coli both at 300 MPa with 1.2 NL/L CO₂ and at 250 MPa with 3.2 NL/L CO₂, while only 2.2 and 1.8 log reductions were observed at 300 MPa and 250 MPa, respectively, for the HHP treatments alone. For S. aureus, the inactivation rate of more than 7 log units was found at 350 MPa with 3.8 NL/L CO₂, while only a 0.9 log reduction was achieved at this pressure in the absence of CO₂. The SEM photographs showed seriously deformed cells after the synergistic treatments. In contrast, the cells treated with individual HHP maintained a relatively smooth surface with invaginations. Propidium iodide staining and fluorescence observation was performed after pressure treatments. The results demonstrated that the combination of CO₂ with HHP also promoted pressure induced cell membrane permeabilization greatly. It was deduced that the enrichment of CO₂ on the cell surface and its penetration into the cells at high pressure accounted for the membrane damage and cell death.     

Statistical optimization,partial purification,and characterization of coffee pulp β-glucosidase and its application in ethanol production

Extracellular β-glucosidase was produced using coffee pulp as a sole carbon source by Penicillium verrucosum by solid state fermentation and 897.36±59 U/g enzyme activity was obtained. Increase in 2.21-fold of enzyme activity on optimizing the bioprocess parameters by response surface methodology based on central composite rotatable design is illustrated. Maximum production level of 1,991.17 U/g was obtained with optimum values of pH 4.2, moisture 66.8%, and fermentation duration of 56 h. The enzyme was partially purified and the enzyme activity was optimum at 50°C temperature and at pH 6. The metal ions such as Mg²⁺, Zn²⁺, Ca²⁺, K⁺, detergents, and chelator such as EDTA were effective and further increased the β-glucosidase activity. On application of β-glucosidase for simultaneous saccharifiation and fermentation, 3.3% ethanol was obtained. Thus, this study provides insight on exploitation of P. verrucosum for synthesis of of β-glucosidase using coffee pulp which is available abundantly in coffee processing industries.

     

Enhancement of β-glucosidase activity on the cell-surface of sake yeast by disruption of SED1

We determined the genetic background that would result in a more optimal display of heterologously expressed β-glucosidase (BGL) on the cell surface of yeast Saccharomyces cerevisiae. Amongst a collection of 28 strains carrying deletions in genes for glycosylphosphatidyl inositol (GPI)-anchored proteins, the Δsed1 and Δtos6 strains had significantly higher BGL-activity whilst maintaining wild type growth. Absence of Sed1p, which might facilitate incorporation of anchored BGL on the cell-surface, could also influence the activity of BGL on the cell surface with the heterologous gene being placed under the control of the SED1 promoter. For the evaluation of its industrial applicability we tested this system in heterologous and homogenous SED1-disruptants of sake yeast, a diploid S. cerevisiae strain, in which either the SED1 ORF or the complete gene including the promoter was deleted by use of the high-efficiency loss of heterozygosity method. Evaluation of disruptants displaying BGL showed that deletion of the SED1 ORF enhanced BGL activity on the cell surface, while additional deletion of the SED1 promoter increased further BGL activity on the cell surface. Compared to heterozygous disruption, homozygous disruption resulted generally in a higher BGL activity. Thus, homozygous deletion of both SED1 gene and promoter resulted in the most efficient display of BGL reaching a 1.6-fold increase of BGL-activity compared to wild type.     

Enhancement of the hydrolysis activity of β-galactosidase from Geobacillus stearothermophilus by saturation mutagenesis

Thermostable β-galactosidase (BgaB) from Geobacillus stearothermophilus is characterized by its thermoactivity in the hydrolysis of lactose to produce lactose-free milk products. However, BgaB has limited activity toward lactose. We established a method for screening evolved mutants with high hydrolysis activity based on prediction of substrate binding sites. Seven amino acid residues were identified as candidates for substrate binding to galactose. To study the hydrolysis activity of these residues, we constructed mutants by site-saturation mutagenesis of these residue sites, and each variant was screened for its hydrolysis activity. The first round of mutagenesis showed that changes in amino acid residues of Arg109, Tyr272, and Glu351 resulted in altered hydrolysis activity, including greater activity toward ortho-nitrophenyl-β-d-galactopyranoside (oNPG). The mutants R109V and R109L displayed changes in the optimum pH from 7.0 to 6.5, and the mutant R109V/L displayed different substrate affinity and catalytic efficiency (k_cat/K_m). Mutant R109G showed complete loss of BgaB enzymatic activity, suggesting that Arg109 plays a significant role in maintaining hydrolysis activity. The optimum pH of mutant E351R increased from 7.0 to 7.5 and this mutant showed a prominent increase in catalytic efficiency with oNPG and lactose as substrates.     

Expression and characterization of β-1,3-1,4-glucanase of Aspergillus usamii in Escherichia coli and its application in sourdough bread making

A β-1,3-1,4-glucanase gene (Auglu12A) from Aspergillus usamii was successfully expressed in Escherichia coli BL21(DE3). The recombinant enzyme, reAuglu12A was efficiently purified using the one-step nickel-nitrilotriacetic acid affinity chromatography. The specific activity of reAuglu12A was 694.8 U/mg, with an optimal temperature of 55°C and pH of 5.0. The reAuglu12A exhibited stability at temperatures up to 60°C and within the pH range of 4.0–5.5. The reAuglu12A hydrolytic activity was increased in the presence of metal ions, especially K⁺ and Na⁺, whereas it exhibited a K_m and V_max of 8.35 mg/mL and 1254.02 µmol/min/mg, respectively, toward barley β-glucan at pH 5.0 and 55°C. The addition of reAuglu12A significantly increased the specific volume (p < 0.05) and reduced crumb firmness and chewiness (p < 0.05) of wheat–barley sourdough bread during a 7-day storage period compared to the control. Overall, the quality of wheat–barley sourdough bread was improved after incorporation of reAuglu12A (especially at 3000 U/300 g). These changes were attributed to the synergistic effect of acidification by sourdough and its metabolites which provided a conducive environment for the optimal action of reAuglu12A in the degradation of β-glucans of barley flour in sourdough. This stabilized the dough structure, thereby enhancing the quality, texture, and shelf life of the bread. These findings suggest that reAuglu12A holds promise as a candidate for β-glucanase application in the baking industry.