Expression and biochemical characterization of a Bacillus subtilis catalase in Pichia pastoris X-33.

Catalase, which catalyzes the decomposition of H2O2 to H2O and O2, is widely used to reduce H2O2 in industrial applications, such as in food processing, textile dyeing and wastewater treatment. In this study, the catalase (KatA) from Bacillus subtilis was cloned and expressed in the yeast Pichia pastoris X-33. The effect of the promoter in the expression plasmid on the activity level of the secreted KatA protein was also studied. First, the gene encoding KatA was cloned and inserted into a plasmid containing an inducible alcohol oxidase 1 promoter (pAOX1) or a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP). The recombinant plasmids were validated by colony PCR and sequencing and then linearized and transformed into the yeast P. pastoris X-33 for expression. With the promoter pAOX1, the maximum yield of KatA in the culture medium reached 338.8 ± 9.6 U/mL in 2 days of shake flask cultivation, which was approximately 2.1-fold greater than the maximum yield obtained with the promoter pGAP. The expressed KatA was then purified from the culture medium by anion exchange chromatography, and its specific activity was determined to be 14826.58 U/mg. Finally, the purified KatA exhibited optimum activity at 25 °C and pH 11.0. Its Km for hydrogen peroxide was 10.9 ± 0.5 mM, and its kcat/Km was 5788.1 ± 25.6 s-1 mM-1. Through the work presented in this article, we have therefore demonstrated efficient expression and purification of KatA in P. pastoris, which might be advantageous for scaling up the production of KatA for use in a variety of biotechnological applications.

Enhanced Extracellular Production and Characterization of Sucrose Isomerase in Bacillus subtilis with Optimized Signal Peptides.

Sucrose isomerase (SIase) catalyzes the hydrolysis and isomerization of sucrose into isomaltulose, which is an important functional sugar widely used in the food industry. However, the lack of safe and efficient expression systems for recombinant SIase has impeded its production and application. In this study, enhanced expression of a SIase from Klebsiella sp. LX3 (referred to as KsLX3-SIase) was achieved in Bacillus subtilis WB800N, by optimizing the signal peptides. First, 13 candidate signal peptides were selected using a semi-rational approach, and their effects on KsLX3-SIase secretion were compared. The signal peptide WapA was most efficient in directing the secretion of KsLX3-SIase into the culture medium, producing a specific activity of 23.0 U/mL, as demonstrated by shake flask culture. Using a fed-batch strategy, the activity of KsLX3-SIase in the culture medium was increased to 125.0 U/mL in a 5-L fermentor. Finally, the expressed KsLX3-SIase was purified and was found to have maximum activity at 45 °C and pH 5.5. Its Km for sucrose was 267.6 ± 18.6 mmol/L, and its kcat/Km was 10.1 ± 0.2 s-1mM-1. These findings demonstrated an efficient expression of SIase in B. subtilis, and this is thought to be the highest level of SIase produced in a food-grade bacteria to date.

[Preparation and catalytic properties of catalase-inorganic hybrid nanoflowers].

Catalase is widely used in the food, medical, and textile industries. It possesses exceptional properties including high catalytic efficiency, high specificity, and environmental friendliness. Free catalase cannot be recycled and reused in industry, resulting in a costly industrial biotransformation process if catalase is used as a core ingredient. Developing a simple, mild, cost-effective, and environmentally friendly approach to immobilize catalase is anticipated to improve its utilization efficiency and enzymatic performance. In this study, the catalase KatA derived from Bacillus subtilis 168 was expressed in Escherichia coli. Following separation and purification, the purified enzyme was prepared as an immobilized enzyme in the form of enzyme-inorganic hybrid nanoflowers, and the enzymatic properties were investigated. The results indicated that the purified KatA was obtained through a three-step procedure that included ethanol precipitation, DEAE anion exchange chromatography, and hydrophobic chromatography. Then, by optimizing the process parameters, a novel KatA/Ca3(PO4)2 hybrid nanoflower was developed. The optimum reaction temperature of the free KatA was determined to be 35 ℃, the optimum reaction temperature of KatA/Ca3(PO4)2 hybrid nanoflowers was 30-35 ℃, and the optimum reaction pH of both was 11.0. The free KatA and KatA/Ca3(PO4)2 hybrid nanoflowers exhibited excellent stability at pH 4.0-11.0 and 25-50 ℃. The KatA/Ca3(PO4)2 hybrid nanoflowers demonstrated increased storage stability than that of the free KatA, maintaining 82% of the original enzymatic activity after 14 d of storage at 4 ℃, whereas the free KatA has only 50% of the original enzymatic activity. In addition, after 5 catalytic reactions, the nanoflower still maintained 55% of its initial enzymatic activity, indicating that it has good operational stability. The Km of the free KatA to the substrate hydrogen peroxide was (8.80±0.42) mmol/L, and the kcat/Km was (13 151.53± 299.19) L/(mmol·s). The Km of the KatA/Ca3(PO4)2 hybrid nanoflowers was (32.75±2.96) mmol/L, and the kcat/Km was (4 550.67±107.51) L/(mmol·s). Compared to the free KatA, the affinity of KatA/Ca3(PO4)2 hybrid nanoflowers to the substrate hydrogen peroxide was decreased, and the catalytic efficiency was also decreased. In summary, this study developed KatA/Ca3(PO4)2 hybrid nanoflowers using Ca2+ as a self-assembly inducer, which enhanced the enzymatic properties and will facilitate the environmentally friendly preparation and widespread application of immobilized catalase.

[Label-free quantitative peptidomics-based analysis on glycopeptides in deerhorn gelatin and deer-hide gelatin].

Nano-LC-MS/MS was used to analyze trypsin digested deer-horn gelatin( DCG) and deer-hide gelatin( DHG) samples.The glycopeptides in DCG and DHG were quantified by Label-free quantitative( LFQ) peptidomics,on the basis of which the glycopeptides with significant difference in DCG and DHG were determined. As a result,5 736 peptides were identified from DCG samples,including 213 galactosyl-hydroxylysine containing peptides( Gal-Hyl-peptides) and 102 glucosyl-galactosyl-hydroxylysine containing peptides( Glc-Gal-Hyl-peptides),while 6 836 peptides were identified from DHG samples,among which there were 250 Gal-Hyl-peptides and 98 Glc-Gal-Hyl-peptides. With over 3-fold peak area difference and highly significant intergroup difference( P < 0. 01) as the screening criteria,444 differential peptides were determined in DCG and DHG,including 16 Gal-Hyl-peptides and 5 Glc-Gal-Hyl-peptides. Then XIC peak shapes,standard deviation of peak area,and fold change were applied for further screening and 5 glycopeptides with significant differences in DCG and DHG were confirmed,which could serve as potential biomarkers for distinguishing DCG and DHG. The present study provided ideas and strategies for the in-depth investigation on the discrimination of DCG and DHG and is of good theoretical significance and application value for the further research on chemical constituents and quality control of gelatin derived Chinese medicinals.

Discovery of peptide biomarkers by label-free peptidomics for discrimination of horn gelatin and hide gelatin from Cervus nippon Temminck.

Gelatin and gelatin-based derivatives have been attracting worldwide attention as health-food ingredients. Deer horn gelatin (DCG), a well-known and expensive gelatin food in Asia, has suffered adulterants by adding deer-hide gelatin (DHG) in it. However, robust and effective methods which could differentiate DCG from DHG are still unavailable. This study is committed to discover peptide biomarkers to distinguish DCG from DHG using label-free peptidomics by nanoLC-MS/MS. Multivariate statistical analysis combined with glycosylation sites analysis of peptides was applied to visualize the difference between DCG and DHG. As a result, four peptide biomarkers for distinguishing DCG and DHG were confirmed and validated by UPLC-MS/MS and MRM mode, which was also used to calculate adulteration percentage in commercial samples. The presented strategy may be also particularly helpful in the in-depth authentication of food gelatins from different tissues of the same species.

[Analysis of hydroxylation and O-glycosylation on lysine sites in deer-hide gelatin].

Nano-LC MS/MS was used to analyze trypsin digested deer-hide gelatin(DHG) samples, hydroxylation and O-glycosylation on lysine sites of DHG were comprehensive identified by using PEAKS Studio software. The sites, sorts and amounts of hydroxylation and O-glycosylation on Type Ⅰ collagen α1 chain(COL1 A1) and α2 chain(COL1 A2) of DHG were revealed. As a result, 5 284 peptides were identified from DHG samples, which were mainly from COL1 A1 and COL1 A2. Among these peptides, there were 449 peptides with hydroxylysine, 442 with galactosyl-hydroxylysine, 449 with glucosyl-galactosyl-hydroxylysine. The major modified sites of hydroxylation and O-glycosylation in DHG were shown as follow: α1-9 N and α2-5 N in N-telopeptides, α1-87, α1-174, α1-930, α2-87, α2-174, α2-933 in triple helix domain, and α1-16 C in C-telopeptides. These hydroxylation and O-glycosylation were correlated with the formation and stability of collagen molecules and collagen fibrils. It is feasible for the collagens and peptides dissolving from deer skin collagen fibrils under high temperature and pressure decocting, high temperature and pressure also might destroy inter-molecular covalent cross-linking and help those glycol-peptides formations. The present study provided ideas and strategies for the in-depth investigation on DHG chemical constituents, and showed good theoretical significance and application value.

Collagen derived species-specific peptides for distinguishing donkey-hide gelatin (Asini Corii Colla).

Objective: As an important food therapy product with traditional Chinese medicine (TCM) applications, donkey-hide gelatin (Asini Corii Colla, ACC) has been used for thousands of years. However, till now few effective strategy had been proposed to distinguish ACC from other animal hide gelatins, especially closely related horse- and mule-hide gelatins, which was an embarrassment of ACC quality control. Methods: Combined mass spectrometry and bioinformatic methods have been applied to identify and verify two ACC-specific peptides (Pep-1 and Pep-2) capable of distinguishing ACC from other closely related animal gelatins with high selectivity. Results: It confirmed that these two peptides could be not only used for distinguishing ACC from highly homologous horse-hide and mule-hide gelatins as well as other animal hide gelatins. Conclusion: The present study provides a simple method for species-specific peptides discovery, which can be used for assessing the quality of animal gelatin products, and ensure they are authenticable and traceable.

Potential ceRNA networks involved in autophagy suppression of pancreatic cancer caused by chloroquine diphosphate: A study based on differentially­expressed circRNAs, lncRNAs, miRNAs and mRNAs.

Autophagy has been reported to be involved in the occurrence and development of pancreatic cancer. However, the mechanism of autophagy­associated non­coding RNAs (ncRNAs) in pancreatic cancer remains largely unknown. In the present study, microarrays were used to detect differential expression of mRNAs, microRNAs (miRNAs), long ncRNAs (lncRNAs) and circular RNAs (circRNAs) post autophagy suppression by chloroquine diphosphate in PANC­1 cells. Collectively, 3,966 mRNAs, 3,184 lncRNAs and 9,420 circRNAs were differentially expressed. Additionally, only two miRNAs (hsa­miR­663a­5p and hsa­miR­154­3p) were underexpressed in the PANC­1 cells in the autophagy­suppression group. Furthermore, miR­663a­5p with 9 circRNAs, 8 lncRNAs and 46 genes could form a prospective ceRNA network associated with autophagy in pancreatic cancer cells. In addition, another ceRNA network containing miR­154­3p, 5 circRNAs, 2 lncRNAs and 11 genes was also constructed. The potential multiple ceRNA, miRNA and mRNA associations may serve pivotal roles in the autophagy of pancreatic cancer cells, which lays the theoretical foundation for subsequent investigations on pancreatic cancer.

Comprehensive analysis of the long noncoding RNA HOXA11-AS gene interaction regulatory network in NSCLC cells.

BACKGROUND: Long noncoding RNAs (lncRNAs) are related to different biological processes in non-small cell lung cancer (NSCLC). However, the possible molecular mechanisms underlying the effects of the long noncoding RNA HOXA11-AS (HOXA11 antisense RNA) in NSCLC are unknown. METHODS: HOXA11-AS was knocked down in the NSCLC A549 cell line and a high throughput microarray assay was applied to detect changes in the gene profiles of the A549 cells. Bioinformatics analyses (gene ontology (GO), pathway, Kyoto Encyclopedia of Genes and Genomes (KEGG), and network analyses) were performed to investigate the potential pathways and networks of the differentially expressed genes. The molecular signatures database (MSigDB) was used to display the expression profiles of these differentially expressed genes. Furthermore, the relationships between the HOXA11-AS, de-regulated genes and clinical NSCLC parameters were verified by using NSCLC patient information from The Cancer Genome Atlas (TCGA) database. In addition, the relationship between HOXA11-AS expression and clinical diagnostic value was analyzed by receiver operating characteristic (ROC) curve. RESULTS: Among the differentially expressed genes, 277 and 80 genes were upregulated and downregulated in NSCLC, respectively (fold change ≥2.0, P < 0.05 and false discovery rate (FDR) < 0.05). According to the degree of the fold change, six upregulated and three downregulated genes were selected for further investigation. Only four genes (RSPO3, ADAMTS8, DMBT1, and DOCK8) were reported to be related with the development or progression of NSCLC based on a PubMed search. Among all possible pathways, three pathways (the PI3K-Akt, TGF-beta and Hippo signaling pathways) were the most likely to be involved in NSCLC development and progression. Furthermore, we found that HOXA11-AS was highly expressed in both lung adenocarcinoma and squamous cell carcinoma based on TCGA database. The ROC curve showed that the area under curve (AUC) of HOXA11-AS was 0.727 (95% CI 0.663-0.790) for lung adenocarcinoma and 0.933 (95% CI 0.906-0.960) for squamous cell carcinoma patients. Additionally, the original data from TCGA verified that ADAMTS8, DMBT1 and DOCK8 were downregulated in both lung adenocarcinoma and squamous cell carcinoma, whereas RSPO3 expression was upregulated in lung adenocarcinoma and downregulated in lung squamous cell carcinoma. For the other five genes (STMN2, SPINK6, TUSC3, LOC100128054, and C8orf22), we found that STMN2, TUSC3 and C8orf22 were upregulated in squamous cell carcinoma and that STMN2 and USC3 were upregulated in lung adenocarcinoma. Furthermore, we compared the correlation between HOXA11-AS and de-regulated genes in NSCLC based on TCGA. The results showed that the HOXA11-AS expression was negatively correlated with DOCK8 in squamous cell carcinoma (r = -0.124, P = 0.048) and lung adenocarcinoma (r = -0.176, P = 0.005). In addition, RSPO3, ADAMTS8 and DOCK8 were related to overall survival and disease-free survival (all P < 0.05) of lung adenocarcinoma patients in TCGA. CONCLUSIONS: Our results showed that the gene profiles were significantly changed after HOXA11-AS knock-down in NSCLC cells. We speculated that HOXA11-AS may play an important role in NSCLC development and progression by regulating the expression of various pathways and genes, especially DOCK8 and TGF-beta pathway. However, the exact mechanism should be verified by functional experiments.

Decoy Receptor 3 (DcR3) as a Biomarker of Tumor Deterioration in Female Reproductive Cancers: A Meta-Analysis.

BACKGROUND DcR3 (decoy receptor 3) has been proposed be involved in development and prognosis of female reproductive cancers, including cervical cancer, ovarian cancer, and breast cancer. The purpose of this meta-analysis was to explore the evidence for the correlation between DcR3 and the clinicopathological characteristics, as well as the overall survival time, in female reproductive cancers. MATERIAL AND METHODS Relevant studies were searched for in PubMed, Wiley Online Library, Web of Science, Science Direct, Cochrane Central Register of Controlled Trials, Google Scholar, EMBASE, Ovid, LILACS, Chinese CNKI, Chong Qing VIP, Wan Fang, and China Biology Medicine disc up to 30 September 2015. Data on the relationship between DcR3 expression and TNM stage, differentiation, lymph node metastasis, age, and overall survival time were extracted. Pooled odds ratios (ORs) and 95% CIs (confidence intervals) were estimated by forest plot. RESULTS Twelve studies with 1127 patients met the inclusion criteria for this meta-analysis. Overexpression of DcR3 was significantly related to the risk of female reproductive cancers (OR=10.69, 95% CI: 6.33-18.05), TNM stage (OR=5.51, 95% CI: 2.83-10.71), differentiation (OR=4.16, 95% CI: 2.28-7.60), lymph node metastasis (OR=5.89, 95% CI: 3.16-10.9), age (OR=0.85, 95% CI: 0.51-1.44), and overall survival time (OR=1.84, 95% CI: 0.58-5.83). Subgroup analyses showed that overexpression of DcR3 in cervical, ovarian, and breast cancer all had similar relationships with these clinicopathological parameters. CONCLUSIONS Our meta-analysis suggests that overexpression of DcR3 may play vital roles in the tumorigenesis and deterioration of female reproductive cancers. However, the relationship between DcR3 expression and prognosis needs further investigation.

Long Noncoding RNA MALAT-1 Can Predict Poor Prognosis: A Meta-Analysis.

BACKGROUND: MALAT-1 is a highly conserved nuclear long non-coding RNA (lncRNA). The overexpression of MALAT-1 has been reported in several types of cancers. This meta-analysis was conducted to further investigate its potential role as a prognostic indicator in various cancers. MATERIAL/METHODS: The meta-analysis was performed by use of systematic search terms in 13 databases for qualified papers on prognosis in cancer from inception to June 30, 2015. The combined hazard ratios (HRs) with 95% confidence interval (95% CI) were computed to demonstrate the effect of MALAT-1 on prognosis of cancers. RESULTS: A total of 590 papers were initially identified, and 17 studies were finally included in this paper. Meta-analysis was accomplished with a total of 1626 patients. Combined HRs and 95% CI were calculated by fixed-effects or random-effects models. The quality assessment of included studies was performed by the Newcastle-Ottawa scale (NOS). High expression of MALAT-1 was found to be an indicator of poor prognosis in overall survival (OS) (HR=1.84, 95% CI: 1.27-2.67) and disease-free survival (DFS) (HR=2.37, 95% CI: 1.55-3.62). In subgroups, the associations between MALAT-1 and survival were also apparent, for instance, in country subgroup: China (HR=1.85, 95% CI: 1.14-2.99). CONCLUSIONS: The overexpression of MALAT-1 may be a potential prognostic indicator for various human cancers.