Construction and secretory expression of beta-galactosidase gene from Lactobacillus bulgaricus in Lactococcus lactis.

OBJECTIVE: This study is to examine the secretion effects of beta-galactosidase in Lactococcus lactis. METHODS: The usp45 and beta-galactosidase genes were cloned and inserted into plasmid pMG36e to obtain the recombinant plasmid pMG36e-usp-lacZ. This recombinant plasmid was transformed into both Escherichia coli DH5alpha and L. lactis MG1363. The enzyme activity, gene sequencing, SDS-PAGE and hereditary stability were assessed and studied. RESULTS: The lacZ gene inserted into plasmids pMG36e-usp-lacZ was 99.37% similar to the GenBank sequence, and SDS-PAGE revealed an evident idio-strap at 116 KDa between L. lactis MG1363/pMG36e-usp-lacZ in both supernatant and cell samples. Beta-Galactosidase activity measured 0.225 U/mL in L. lactis pMG36e-usp-lacZ transformants, and its secretion rate was 10%. The plasmid pMG36e-usp-lacZ appeared more stable in MG1363. CONCLUSION: The authors concluded that these new recombinant bacteria well expressed and secreted beta-galactosidase, indicating that the beta-galactosidase expression system was successfully constructed, and this might provide a new solution for management of lactose intolerance specifically and promote the use of gene-modified organisms as part of the food-grade plasmid in general.

Analysis of ß-galactosidase production and their genes of two strains of Lactobacillus bulgaricus.

A bacterial ß-galactosidase delivery system is a potential therapy for lactose intolerance. Currently, two Lactobacillus bulgaricus strains with different biological characteristics are under consideration as potential sources. However, differences in these ß-galactosidase genes and their resulting production levels are poorly characterized. The ß-galactosidase ORF of L. bulgaricus yogurt isolate had high variability and was terminated at site 1924 due to a stop codon. However, the full 114 kDa ß-galactosidase band was still resolved by SDS-PAGE, which may indicate that the interrupted ORF was translated into more than one peptide, and they together were folded into the complete enzyme protein that showed much higher ß-galactosidase activity (6.2 U/mg protein) than the enzyme generated from L. bulgaricus reference strain (2.5 U/mg protein).

[Construction and property study of recombinant Lactococcus lactis with non-fusion expressing of beta-galactosidase].

OBJECTIVE: To construct recombinant Lactococcus lactis strains exhibiting high beta-galactosidase activity in non-fusion way, and study their enzyme activities and enzyme secretion rates. METHODS: The recombinant plasmids pMG36e-lacZ 1.1480 and pMG36e-lacZ wch9901 which could express beta-galactosidase from Lactobacillus delbrueckii subsp. bulgaricus in non-fusion way in Escherichia coli were obtained and transformed into Lactococcus lactis subsp. lactis MG1363. The beta-galactosidase activity of resulting recombinant L. lactis in different incubation periods and lactose concentrations, and their enzyme secretion rates in different culture conditions were examined. RESULTS: Recombinant L. lactis carrying pMG36e-lacZ wch9901 (MG1363/pMG36e-lacZ wch9901) exhibited the highest beta-galactosidase activity. Its enzyme activity was (16.95 +/- 0.09) U/mg pro, which was 2.75 folds of that of the native counterpart; recombinant L. lactis reached its enzyme producing peak after grown for 24 h; decreased enzyme activity of recombinant L. lactis were observed when incubated in medium containing lactose; the beta-galactosidase expressed by recombinant strains could be secreted into the culture medium, and the highest secretion rate (27.09 +/- 0.05)% was observed when the culture medium contained 20 g/L of lactose and without erythromycin. CONCLUSION: High level expression of non-fusion beta-galactosidase with secretion in recombinant L. lactis strains was obtained. This will be very helpful for the further developing of live delivery bacteria of beta-galactosidase.

[Beta-galactosidase gene from Lactobacillus delbrueckii subsp. bulgaricus gets non-fusion expression in Escherichia coli].

OBJECTIVE: To construct the food-grade recombinant probiotic strain with high activity beta-galactosidase, the beta-galactosidase gene (lacZ)from Lactobacillus delbrueckii subsp. bulgaricus was in non-fusion expressed in Escherichia coli. METHODS: From Lb. delbrueckii subsp. bulgaricus lacZ gene, the DNA sequence containing Shine-Dalgarno (SD) and ATGA sequences between upstream 18 bp and downstream 1 bp at start codon ATG was selected as upstream primer for PCR amplifying lacZ gene. Then lacZ cDNA was inserted into expression plasmid pMG36e to construct recombinant expression plasmid. Recombinant plasmids were introduced into E. coli, and positive clones were screened. To identify the gene recombination, the recombinant plasmid was cut by restriction enzyme and sequenced. To identify the protein expression, the beta-galactosidase activities of recombinant strains were determined. RESULTS: The restriction maps of recombinant plasmids were acceptable. The gene inserted into the recombinant plasmid had more than 99% homology with the lacZ gene of Lb. delbrueckii subsp. bulgaricus. The enzyme activity and enzyme activity ratio of E. coli DH5 alpha carrying pMG36e-lacZ 1.1480 were 3.074 U/mL and 6.939 U/mg pro respectively. The enzyme activity and enzyme activity ratio of E. coli DH5a carrying pMG36e-lacZ wch9901 were 4.755 U/mL and 8.537 U/mg pro respectively. CONCLUSION: lacZ from Lb. delbrueckii subsp. bulgaricus have gotten non-fusion expression in E. coli. The SD and ATGA sequences we selected can introduce lacZ non-fusion expression in E. coli.

[Phylogeny analysis and identification of two bacterial strains sourcing from human intestine and having resistance to acid and bile].

OBJECTIVE: To screen bacteria for the engineering bacteria expressing and secreting high activity beta-galactosidase, and two bacterial strains called as R92-2 and R111 with acid and bile resistances would be isolated from health human intestine to strain identification and phylogenetic analysis. METHODS: These two strains were first been identified with phenotype characteristic analysis. Then the 16S rDNAs of these two bacterial strains were amplified and sequenced with the primers designed by the conserve sequences. DNA sequences were blasted against GenBank, and the phylogenetic trees were constructed. RESULTS: Phenotype characteristic analysis showed that both of the bacterial strains belonged to lactic acid bacteria. Results of Blastn showed that 16S rDNAs of R92-2 and Weissella cibaria had 100% homology; 16S rDNAs of R111 and Enterococcus faecium had 100% homology too. CONCLUSION: R92-2 belongs to Weissella cibaria strain; R111 belongs to Enterococcus faecium strain. These two stains may be used to construct the engineering bacteria expressing and secreting high activity beta-galactosidase.

[Comparative study of the resistance of bacteriophageT4, PhiX174D, MS2 and f2 to gamma radiation].

OBJECTIVE: To screen the suitable bacteriophage as virus indicator in irradiation sterilization. METHODS: Suspensions of bacteriophage T4, phiX174D, MS2, and f2, Escherichia coli 8099, and Bacillus subtilis var.niger.sp. ATCC9372 were irradiated with (60)Co-gamma ray. The mean log(10) inactivation value (LIV) and killing log value (KL) were calculated. RESULTS: (1) Under 100 Gy of gamma-radiation, the LIV levels of the bacteriophage T4, PhiX174, f2, and MS2 were 6.31, 6.92, 5.74, and 4.46 log(10) respectively, all reaching the disinfection level (LIV >/= 4.00 log(10)), (2) Under the same absorbed dose, the KL of Escherichia coli 8099 was > 7.97 log(10); (3) Under the same absorbed dose, the KL of the Bacillus subtilis var.niger.sp. ATCC9372 was 1.61 log(10). CONCLUSION: The order of resistance of the above six microorganisms to gamma-radiation from the biggest to the smallest is as follows: Bacillus subtilis var. niger. sp. > bacteriophage MS2 > bacteriophage f2 > bacteriophage T4 > bacteriophage phiX 174D > E. coli.

Non-fusion and fusion expression of beta-galactosidase from Lactobacillus bulgaricus in Lactococcus lactis.

OBJECTIVE: To construct four recombinant Lactococcus lactis strains exhibiting high beta-galactosidase activity in fusion or non-fusion ways, and to study the influence factors for their protein expression and secretion. METHODS: The gene fragments encoding beta-galactosidase from two strains of Lactobacillus bulgaricus, wch9901 isolated from yogurt and 1.1480 purchased from the Chinese Academy of Sciences, were amplified and inserted into lactococcal expression vector pMG36e. For fusion expression, the open reading frame of the beta-galactosidase gene was amplified, while for non-fusion expression, the open reading frame of the beta-galactosidase gene was amplified with its native Shine-Dalgarno sequence upstream. The start codon of the beta-galactosidase gene partially overlapped with the stop codon of vector origin open reading frame. Then, the recombinant plasmids were transformed into Escherichia coli DH5 alpha and Lactococcus lactis subsp. lactis MG1363 and confirmed by determining beta-galactosidase activities. RESULTS: The non-fusion expression plasmids showed a significantly higher beta-galactosidase activity in transformed strains than the fusion expression plasmids. The highest enzyme activity was observed in Lactococcus lactis transformed with the non-fusion expression plasmids which were inserted into the beta-galactosidase gene from Lactobacillus bulgaricus wch9901. The beta-galactosidase activity was 2.75 times as high as that of the native counterpart. In addition, beta-galactosidase expressed by recombinant plasmids in Lactococcus lactis could be secreted into the culture medium. The highest secretion rate (27.1%) was observed when the culture medium contained 20 g/L of lactose. CONCLUSION: Different properties of the native bacteria may have some effects on the protein expression of recombinant plasmids. Non-fusion expression shows a higher enzyme activity in host bacteria. There may be a host-related weak secretion signal peptide gene within the structure gene of Lb. bulgaricus beta-galactosidase, and its translation product may introduce the enzyme secretion out of cells in special hosts.

[Comparative study on the resistance of Bacteriophage phi chi 174D, T4 and f2 to an iodophor in laboratory].

BACKGROUND: To screen for the most resistant bacteriophage as indicator in disinfection tests, the resistance of bacteriophage phi chi 174D, T4 and f2 to iodophor were observed in laboratory. METHODS: The virucidal activity of iodophor against bacteriophage phi chi 174D, T4, and f2 were assessed by suspension test. The neutralizer is selected and appraised by testing with neutralizer. Bacteriophage phi chi 174D, T4, and f2 were detected and enumerated by the double-agar-layer plaque technique. RESULTS: (1) With 500 mg/L of available iodine of iodophor solution, within a contact time of 40 min, or 750 mg/L, 10 min, or 1000 mg/L, 5 min, the reduction of bacteriophage phi chi 174D could achieve the "disinfection" level [log10 inactivation value (LIV) or log10 reduction value (LRV) of bacteriophage phi chi 174D (log10 No-log10 Nt) was > or = 4.00 log10]. (2) With 600 mg/L of available iodine of iodophor solution, within a contact time of 40 min, or 700 mg/L, 5 min, the reductions of bacteriophage T4 could achieve the "disinfection" level. (3) With 50 mg/L of available iodine of iodophor solution, within a contact time of 10 min, or 75 mg/L, 10 min, the reductions of bacteriophage f2 could achieve the "disinfection" level. CONCLUSION: The order of resistance of the above three bacteriophages to iodophor from greatest to smallest is as follows: bacteriophage phi chi 174D greater than bacteriophage T4 > bacteriophage f2.

[Comparison on resistance of bacteriophages to sodium dichloroisocyanurate in laboratory].

OBJECTIVE: To scan the most resistable bacteriophage as an indicator in disinfection tests, and to study the resistance of bacteriophage T4, Phichi 174D, and f2 to the sodium dichloroisocyanurate (NaDCC) in laboratory. METHODS: The virucidal activity of NaDCC against bacteriophage T4, Phichi 174D, and f2 were assessed by suspension test. The neutralizer was selected and be appraised by test of neutralizer. Bacteriophage T4, Phichi 174D, and f2 were detected and enumerated by the double-agar-layer plaque technique. RESULTS: (1) With 150 mg/L of available chlorine of NaDCC solution, within a contact time of 40 minutes, or 300 mg/L, 5 minutes, the reductions of bacteriophage T4 achieved the "disinfection" level [log(10) inactivation value or log(10) reduction value of bacteriophage T4 (log(10)No-log(10)Nt) > or = 4.00 log(10)]. (2) With 300 mg/L of available chlorine of NaDCC solution, within a contact time of 5 minutes, or 400 mg/L, 3 minutes, the reductions of bacteriophage Phichi 174D achieved the "disinfection" level. (3) With 2000 mg/L of available chlorine of NaDCC solution, within a contact time of 20 minutes, or 4000 mg/L, 5 minutes, the reductions of bacteriophage f2 might achieve the "disinfection" level. CONCLUSION: The order of resistance of the above three bacteriophages to NaDCC from greatest to smallest is as follows: bacteriophage f2 > bacteriophage T4 > bacteriophage Phichi 174D.

[Construction and expression of Neisseria surface protein (nspA) of Neisseria gonorrhoeae in Escherichia coli].

OBJECTIVE: To construct neisseria surface protein (NspA) recombinants of Neisseria gonorrhoeae from a reference strain and express this protein in E. coli. METHODS: The fragments of NspA gene of Neisseria gonorrhoeae was amplified by PCR from the reference strain genomic DNA and cloned into expression vector pET-30c (+) to get the pET-NspA recombinants. The recombinants were verified with restrictive endonuclease digestion and sequence analysis. The verified recombinant was transformed into E. coli BL21 (DE3). After inducing with IPTG, the expressed NspA protein was analyzed by SDS-PAGE and Western Blot. RESULTS: The pET-NspA expression recombinants for the reference strain of Neisseria gonorrhoeae were successfully constructed and the induced recombinant NspA protein was observed. CONCLUSION: The successful expression of the Neisseria gonorrhoeae NspA protein will be very helpful for the further research of its antigenicity and immunological activity, and for the construction of preventive vaccines on Neisseria gonorrhoeae infection.

[A comparative study on the resistance of bacteriophage T4, phiXM174D, and f2 to glutaraldehyde in laboratory].

OBJECTIVE: To identify the best indicative bacteriophage in disinfection tests through comparing the resistance of bacteriophage T4, phiX174D, and f2 to glutaraldehyde. METHODS: The virucidal activities of glutaraldehyde against bacteriophage T4, phiX174D, and f2 were assessed with suspension tests along with neutralizer tests. The double-agar-layer plaque technique was used to detect the bacteriophage T4, phiX174D, and f2. RESULTS: (1)In a condition of 3000 mg/L of glutaraldehyde and 20 minutes of contact or 6000 mg/L of glutaraldehyde and 5 minutes of contact, "disinfection" level for bacteriophage T4 was achieved, with the log10 inactivation value (LIV) or log10o reduction value (LRV) (=log10No-log10Nt) > or = 4. 00 log10. (2) In a condition of 2500 mg/L of glutaraldehyde and 20 minutes of contact or 5000 mg/L of glutaraldehyde and 5 minutes of contact, the LIV for bacteriophage phiX174D reached "disinfection" level; (3) In a condition of 4000 mg/L of glutaraldehyde and 40 minutes of contact or 8000 mg/L of glutaraldehyde and 10 minutes of contact, the LIV for bacteriophage f2 reached "disinfection" level. CONCLUSION: Bacteriophage f2 and bacteriophage phiX174D has the strongest and weakest resistance to glutaraldehyde respectively.

[Relief effect of beta-galactosidase genetically engineered lactococcus lactis on the cell toxicity caused by lactose].

OBJECTIVE: To assess the relief effect of beta-galactosidase genetically engineered Lactococcus lactis on the cell toxicity caused by lactose in vitro. METHODS: An in vitro toxic Caco-2 cell model caused by lactose was established to evaluate the relief effect of beta-galactosidase genetically engineered Lactococcus lactis. Cell morphological parameters and proliferation activity parameter were used. RESULTS: The in vitro toxic Caco-2 cell model caused by lactose was successfully established; the genetically engineered Lactococcus lactis constructed in the authors' laboratory could enable the Caco-2 cell to have normal appearance with the presence of lactose and could improve the proliferation activity with the presence of high concentration of lactose (P < 0.01). CONCLUSION: The beta-galactosidase genetically engineered Lactococcus lactis has significant relief effect on the cell toxicity caused by lactose in vitro, which lays a foundation for food-grade alternation of this bacterium.

Comparison of susceptibilities of M. tuberculosis H37Ra and M. chelonei subsp. abscessus to disinfectants.

OBJECTIVE: To determine the susceptibilities of M. tuberculosis H37Ra and M. chelonei subsp. absecessus to several frequently-used disinfectants and to evaluate the practicability of surrogating M. tuberculosis by the latter. METHODS: A suspension quantitative bactericidal test was set up in accordance with Chinese Technique Standard for Disinfection to evaluate the susceptibility of each mycobacteria strain to each selected disinfectant. Killing log value was used as criterion in comparing the susceptibility to disinfectants between the two strains. RESULTS: M. chelonei subsp. abscessus was more resistant to chlorine disinfectant than M. tuberculosis while the two strains were similarly resistant to iodophor disinfectant, peracetic acid, alcohol and glutaraldehyde disinfectant. CONCLUSION: M. chelonei subsp. abscessus has the potential to surrogate M. tuberculosis in evaluating mycobactericidal efficacies of disinfectants.

[Study of the genetic stability of expression plasmid vector pMG36e in host bacteria].

OBJECTIVE: To study the genetic stability of expression plasmid vector pMG36e in Escherichia coli JM109 and Lactococcus lactis MG1363/36e and observe the effect of erythomycin for its stability. METHODS: We used classical method to determine the genetic stable rates of pMG36e in E. coli JM109 and Lactococcus lactis MG1363/36e with or without erythomycin selective pressure. RESULTS: When E. coli JM109 were continuously incubated to 120 generations, its pMG36e genetic stable rates were 100% and 96% respectively with or without erythomycin selective pressure. When Lactococcus lactis MG1363/36e were continuously incubated to 100 generations, its pMG36e genetic stable rates were the same 98%, with or without erythomycin selective pressure. CONCLUSION: pMG36e had good genetic stability in this two hosts. Erythromycin selective pressure had no significant effect for the genetic stability of pMG36e.

[Quantitative analysis of dominating intestinal flora among groups of people with different body fat].

OBJECTIVE: To investigate the alteration of dominating intestinal floras among groups of people with different body fat and probe into the possible effect on lipid metabolism. METHODS: According to the BMI values, subjects were divided into 4 groups, including fleshless group, normal group, overweight group and obese group. Five dominating floras of all fresh stools were quantitated using selective culture method, and all data were analyzed statistically. RESULTS: With the increase of BMI values, there is a decreasing trend in the amount of Lactobacillus, Bifidobacterium and Enterobacillus, and the amounts of Bacteriodes obviously increased (P < 0.01). No obvious alternation of the amounts of Enterococcus and Clostridium was observed. CONCLUSION: Bacteriodes may exert an boosting effect on the pile of fat and development of obese, however Enterobacillus, Lactobacillus, and Bifidobacterium have a potential opposite effect.

[A follow-up study on dietary factors of kidney stones in Bao'an district, Shenzhen City].

OBJECTIVE: To evaluate the major dietary factors of kidney stones in Bao'an District of Shenzhen City and provide a scientific base for further effective prevention of kidney stones. METHODS: Following the process of stratified cluster random sampling in Bao'an district, a cross-sectional study (July-Aug, 2000) was conducted for collecting the base-line data on kidney stones from a population of permanent residents who were over 15 years old, exclusive of those who had had kidney stones or could not correctly respond to the questionnaire review. Then, a follow-up survey (July-Sept, 2002) for incident kidney stone cases was carried out among those residents. The methods for measurement included questionnaire and face-to-face interview, clinical examination and abdominal ultrasonography. All the investigators and interviewers were trained for the field work. And the data processing, dataset and analyses were performed using Visual-Fox 6.0 and SAS 6.12. The risk factors of kidney stoned were comprehensively analyzed for dietary, life style, and family history of stones. The statistical analyses included case-control comparison, factor analysis, correlation analysis, cluster analysis and logistic regression. RESULTS: There were 305 kidney stones patients among 4552 follow-up members, the cumulative incidence of 2 years was 6.92%. The kidney stones were associated with the factors: menopause, RR=2.433; family history of stones, RR=1.544; sea foods, the RR (5-7 times/week vs < or = 1-2 times/month) was 9.032; fruits, the RR (< or = 1-2 times/month vs > or = 1-2 times/week) was 2. 249; sweet foods, the RR (5-7 times/week vs 1-2 times/week) was 2. 568; bean and bean products, the RR (5-7 times/week and < or = 1-2 times/month vs 1-2 times/week) was 2.184 and 1.689. CONCLUSION: Changing the inappropriate habitual eating patterns and generalizing the use of proportioning dietary should be the main measures to prevent kidney stones.

[Amplification of Lactobacillus bulgaricus beta-galactosidase gene and optimization of the reaction condition].

OBJECTIVE: To obtain the gene which encode high activity beta-galactosidase from Lactobacillus bulgaricus and study the influencing factors of amplification. METHODS: Lysozyme and freeze-thaw cycles were applied to obtain Lactobacillus bulgaricus DNA; different template concentration and annealing temperature were selected for the amplification. RESULTS: 60 ng/microliter template concentration and 66 degrees C annealing temperature were the best reaction conditions of amplifying the gene. CONCLUSION: This study optimized the template concentration and anneal temperature conditions of amplifying Lactobacillus bulgaricus beta-galactosidase gene, thus providing a foundation for further studies.