Structural Conversion from Non-native to Native form of Recombinant Human Epidermal Growth Factor by Brevibacillus choshinensis

Brevibacillus choshinensis (Bacillus brevis) HPD31 is a very efficient producer of recombinant human epidermal growth factor (EGF). The produced EGF is secreted into the medium with high efficiency. However part of the EGF that accumulates in the medium, exists as multimeric forms which are biologically inactive. We found the bacterium has the activity to structurally convert multimeric forms to the monomeric, native ones. Optimal temperature and pH for the conversion were 40°C and pH 9, respectively. The reaction was promoted in the presence of reduced glutathione or cysteine. But the cells which had been sonicated or exposed to moderate heat treatment completely lost the activity. Thus, it was presumed that the activity might be due to the enzyme(s) that catalyze the protein disulfide exchanging reaction, and that they resides on the surface of viable cells.     

Sterol Composition in the Pollens of Citrus unshiu Marcov. and Citrus sinensis Osbeck

Bacillus brevis secretes a large amount of cell wall proteins into the culture medium. For construction of Bacillus brevis expression-secretion vectors of human erythropoietin (EPO) and the extracellular domain of mouse erythropoietin receptor (sEPOR), cDNA for each mature form was inserted into a plasmid containing the promoter region and the signal-peptide encoding region of a cell wall protein. Culture supernatants of transformants were affinity purified using a monoclonal antibody-fixed gel for EPO and an EPO-fixed gel for sEPOR. The affinity purification efficiently removed unwanted proteins, giving samples with sufficiently high purity to analyze amino acid sequences of N-terminal regions and biological activities. Combination of this secretory production and affinity purification may facilitate isolation of a large amount of pure EPO and sEPOR, and is useful for further understanding the molecular mechanism of interaction between EPO and EPOR.     

Optimization of Exopolysaccharide Overproduction by Lactobacillus confusus in Solid State Fermentation under High Salinity Stress

It is believed that high concentrations of sodium chloride (NaCl) suppress the biosynthesis of exopolysaccharide (EPS) in lactic acid bacteria (LAB). Nevertheless, overproduction of EPSs due to high salinity stress in solid state fermentation performed on an agar surface was demonstrated in this study using a response surface methodology via a central composite design (CCD). Under optimized conditions with NaCl 4.97% and sucrose 136.5 g/L at 40.79 h of incubation, the EPS yield was 259% (86.36 g/L of EPS), higher than the maximum yield produced with the modified MRS medium containing only 120 g/L of sucrose without NaCl (33.4 g/L of EPS). Biosynthesis of EPS by Lactobacillus confusus TISTR 1498 was independent of biomass production. Our results indicated that high salinity stress can enhance EPS production in solid state fermentation.     

Molecular cloning,characterization and comparison of bile salt hydrolases from Lactobacillus johnsonii PF01

Aims

To clone, characterize and compare the bile salt hydrolase (BSH) genes of Lactobacillus johnsonii PF01.

Methods and Results

The BSH genes were amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers, and the products were inserted into the pET21b expression vector. Escherichia coli BLR (DE3) cells were transformed with pET21b vectors containing the BSH genes and induced using 0·1 mmol l^?1 isopropylthiolgalactopyranoside. The overexpressed BSH enzymes were purified using a nickel–nitrilotriacetic acid (Ni²⁺‐NTA) agarose column and their activities characterized. BSH A hydrolysed tauro‐conjugated bile salts optimally at pH 5·0 and 55°C, whereas BSH C hydrolysed glyco‐conjugated bile salts optimally at pH 5·0 and 70°C. The enzymes had no preferential activities towards a specific cholyl moiety.

Conclusions

BSH enzymes vary in their substrate specificities and characteristics to broaden its activity. Despite the lack of conservation in their putative substrate‐binding sites, these remain functional through motif conservation.

Significance and Impact of the Study

This is to our knowledge the first report of isolation of BSH enzymes from a single strain, showing hydrolase activity towards either glyco‐conjugated or tauro‐conjugated bile salts. Future structural homology studies and site‐directed mutagenesis of sites associated with substrate specificity may elucidate specificities of BSH enzymes.     

Isolation and characterization of bacteriocin‐producing bacteria from the intestinal microbiota of elderly Irish subjects

Aims

To isolate and characterize bacteriocins produced by predominant species of lactic acid bacteria from faeces of elderly subjects.

Methods and Results

Screening over 70 000 colonies, from faecal samples collected from 266 subjects, using the indicator organisms Lactobacillus bulgaricus LMG 6901 and Listeria innocua DPC 3572, identified 55 antimicrobial‐producing bacteria. Genomic fingerprinting following ApaI digestion revealed 15 distinct strains. The antimicrobial activities associated with 13 of the 15 strains were sensitive to protease treatment. The predominant antimicrobial‐producing species were identified as Lactobacillus salivarius, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus crispatus and Enterococcus spp. A number of previously characterized bacteriocins, including ABP‐118 and salivaricin B (from Lact. salivarius), enterocin B (Enterococcus faecium), lactacin B (Lact. acidophilus), gassericin T and a variant of gassericin A (Lact. gasseri), were identified. Interestingly, two antimicrobial‐producing species, not generally associated with intestinally derived microorganisms were also isolated: Lactococcus lactis producing nisin Z and Streptococcus mutans producing mutacin II.

Conclusion

These data suggest that bacteriocin production by intestinal isolates against our chosen targets under the screening conditions used was not frequent (0·08%).

Significance and Impact of the Study

The results presented are important due to growing evidence indicating bacteriocin production as a potential probiotic trait by virtue of strain dominance and/or pathogen inhibition in the mammalian intestine.     

儿童肠道双歧杆菌和乳杆菌种群结构分析

以21例2～5岁中国儿童的肠道菌群为研究对象,利用传统培养计数法和分子生物学技术,对此年龄段健康儿童的肠道菌群分布及其中关键益生菌的种群结构进行了定量研究。实验表明,儿童肠道厌氧菌的数量高达109CFUg(湿重),其肠道菌群的定植抗力(平均BE=2.38)较强;不同的个体之间所能检测到的关键益生菌的种类有所不同,一般能检测到其中的1～4种双歧杆菌和1～5种乳杆菌;长双歧杆菌和假小链双歧杆菌的平均数量多达107CFUg(湿重),检出率分别为90.48%和85.71%,为儿童肠道内双歧杆菌的优势菌种;L.mucosae和发酵乳杆菌的数量较多,平均为3.68log10CFUg(湿重)和3.97log10CFUg(湿重),检出率分别为71.43%和52.38%,为稳定定植于儿童肠道内的优势乳杆菌;不同种类的益生菌在不同样本之间的数量组成均存在有很大差异,双歧杆菌的样本差异为1.86～3.85,乳杆菌的为2.43～4.07。     

Improved production of 3‐hydroxypropionaldehyde by complex formation with bisulfite during biotransformation of glycerol

3‐Hydroxypropionaldehyde (3HPA) is an important specialty chemical which can be produced from glycerol using resting cells of Lactobacillus reuteri. This biocatalytic route, however, suffers from substrate‐ and product‐mediated loss of enzyme activity within 2 h of biotransformation. In order to overcome the inhibitory effects of 3HPA, complex formation with sodium bisulfite was investigated, optimized and applied for in situ capture of the aldehyde during biotransformation of glycerol in a fed‐batch process. As a result, the activity of the cells was maintained for at least 18 h. The 3HPA produced per gram cell dry weight was increased 5.7 times compared to the batch production process, and 2.2 times compared to fed‐batch process without in situ complex formation. This approach may have potential for production and in situ removal of 3HPA after further process development. Biotechnol. Bioeng. 2013; 110: 1243–1248. © 2012 Wiley Periodicals, Inc.     

A Novel Type of Peptidoglycan-binding Domain Highly Specific for Amidated d-Asp Cross-bridge,Identified in Lactobacillus casei Bacteriophage Endolysins

Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-l-alanine amidase, whereas Lc-Lys-2 is a γ-d-glutamyl-l-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with d-Ala⁴→d-Asx-l-Lys³ in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting d-Ala⁴→l-Ala-(l-Ala/l-Ser)-l-Lys³; moreover, they do not lyse the L. lactis mutant containing only the nonamidated d-Asp cross-bridge, i.e. d-Ala⁴→d-Asp-l-Lys³. In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 l-Lys³-d-Asn-l-Lys³ bridges replacing the wild-type 4→3 d-Ala⁴-d-Asn-l-Lys³ bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly d-Asn but not PG with only the nonamidated d-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the d-Asn interpeptide bridge of PG.