Enzymatic Synthesis of Noncanonical α-Amino Acids Containing γ-Tertiary Alcohols.

Noncanonical amino acids (ncAAs) containing tertiary alcohols are valuable as precursors of natural products and active pharmaceutical ingredients. However, the assembly of such ncAA scaffolds from simple material by C-C bond formation remains a challenging task due to the presence of multiple stereocenters and large steric hindrance. In this study, we present a novel solution to this problem through highly selective enzymatic decarboxylative aldol addition. This method allows for the streamlined assembly of multifunctionalized ncAAs with γ-tertiary alcohols from readily available materials, such as L -aspartatic acid and isatins, vicinal diones and keto esters. The modularity of electrophiles furnished four classes of ncAAs with decent efficiency as well as excellent site and stereocontrol. Computational modeling was employed to gain detailed insight into the catalytic mechanism and to provide a rationale for the observed selectivities. The method offers a single-step approach to producing multifunctionalized ncAAs, which can be directly utilized in peptide synthesis and bioactivity assessment.

Functional analysis of the OsNPF4.5 nitrate transporter reveals a conserved mycorrhizal pathway of nitrogen acquisition in plants.

Low availability of nitrogen (N) is often a major limiting factor to crop yield in most nutrient-poor soils. Arbuscular mycorrhizal (AM) fungi are beneficial symbionts of most land plants that enhance plant nutrient uptake, particularly of phosphate. A growing number of reports point to the substantially increased N accumulation in many mycorrhizal plants; however, the contribution of AM symbiosis to plant N nutrition and the mechanisms underlying the AM-mediated N acquisition are still in the early stages of being understood. Here, we report that inoculation with AM fungus Rhizophagus irregularis remarkably promoted rice (Oryza sativa) growth and N acquisition, and about 42% of the overall N acquired by rice roots could be delivered via the symbiotic route under N-NO3- supply condition. Mycorrhizal colonization strongly induced expression of the putative nitrate transporter gene OsNPF4.5 in rice roots, and its orthologs ZmNPF4.5 in Zea mays and SbNPF4.5 in Sorghum bicolor OsNPF4.5 is exclusively expressed in the cells containing arbuscules and displayed a low-affinity NO3- transport activity when expressed in Xenopus laevis oocytes. Moreover, knockout of OsNPF4.5 resulted in a 45% decrease in symbiotic N uptake and a significant reduction in arbuscule incidence when NO3- was supplied as an N source. Based on our results, we propose that the NPF4.5 plays a key role in mycorrhizal NO3- acquisition, a symbiotic N uptake route that might be highly conserved in gramineous species.

Auxin-mediated regulation of arbuscular mycorrhizal symbiosis: A role of SlGH3.4 in tomato.

Most land plants can establish symbiosis with arbuscular mycorrhizal (AM) fungi to increase fitness to environmental challenges. The development of AM symbiosis is controlled by intricate procedures involving all phytohormones. However, the mechanisms underlying the auxin-mediated regulation of AM symbiosis remains largely unknown. Here, we report that AM colonisation promotes auxin response and indole-3-acetic acid (IAA) accumulation, but downregulates IAA biosynthesis genes in tomato (Solanum lycopersicum). External IAA application modulates the AM symbiosis by promoting arbuscule formation at low concentrations but repressing it at high concentrations. An AM-induced GH3 gene, SlGH3.4, encoding a putative IAA-amido synthetase, negatively regulates mycorrhization via maintaining cellular auxin homoeostasis. Loss of SlGH3.4 function increased free IAA content and arbuscule incidence, while constitutively overexpressing SlGH3.4 in either tomato or rice resulted in decreased IAA content, total colonisation level and arbuscule abundance in mycorrhizal roots. Several auxin-inducible expansin genes involved in AM formation or resistance to pathogen infection were upregulated in slgh3.4 mycorrhizal roots but downregulated in the SlGH3.4-overexpressing plants. Taken together, our results highlight a positive correlation between the endogenous IAA content and mycorrhization level, particularly arbuscule incidence, and suggest that the SlGH3.4-mediated auxin homoeostasis and regulation of expansin genes is involved in finely tuning the AM development.

Integrative, genome-wide association study identifies chemicals associated with common women's malignancies.

BACKGROUND: Breast cancer, cervical cancer, and ovarian cancer are three of the most commonly diagnosed malignancies in women, and more cancer prevention research is urgently needed. METHODS: Summary data of a large genome-wide association study of female cancers were derived from the UK biobank. We performed a transcriptome-wide association study and a gene set enrichment analysis to identify correlations between chemical exposure and aberrant expression, repression, or mutation of genes related to cancer using the Comparative Toxicogenomics Database. RESULTS: We identified five chemicals (NSC668394, glafenine, methylnitronitrosoguanidine, fenofibrate, and methylparaben) that were associated with the incidence of both breast cancer and cervical cancer. CONCLUSION: Using a transcriptome-wide association study and gene set enrichment analysis we identified environmental chemicals that are associated with an increased risk of breast cancer, cervical cancer, and ovarian cancer.

Efficacy and safety of cord blood-derived cytokine-induced killer cells in treatment of patients with malignancies.

BACKGROUND AIMS: Adoptive immunotherapy with the use of cytokine-induced killer (CIK) cells represents an effective therapeutic option for treating malignancies. The characteristics and function of cord blood-derived CIK (CB-CIK) cells have been evaluated both in vitro and in vivo. In this study, we assessed the efficacy and safety of administering CB-CIK cells to patients with cancer. METHODS: In this retrospective clinical trial, 15 patients with cancer received CB-CIK therapy with different cycles from April 2012 to August 2014. CB-CIK cells demonstrated a high percentage of main functional fraction CD3(+)CD56(+) and efficient anti-tumor activity in vitro. RESULTS: After the infusion of CB-CIK cells, the subsets of CD3(+)CD4(+) T lymphocytes and CD3(-)CD56(+) T cells in the peripheral blood were significantly increased compared with those before the therapy. Of 15 patients, one patient with hepatocellular cancer and one patient with esophageal cancer achieved complete responses, two patients with ovarian cancer obtained partial remissions, 10 patients had stable disease and one patient with hepatocellular cancer had progressive disease. Acute toxicities including fever, slight fever, dizziness and other neurologic toxicities were few and occurred in patients after infusion of CB-CIK cells. CONCLUSIONS: These results demonstrated the feasibility and safety of treating malignancies with CB-CIK cells. The study provides a potential therapeutic approach for the patients with poor health or older patients who cannot tolerate repeated collection of blood.

Ratiometric fluorescence and absorbance dual-model immunoassay based on 2,3-diaminophenazine and carbon dots for detecting Aflatoxin B1.

In this work, a dual-model immunoassay for detecting Aflatoxin B1 (AFB1) was developed based on 2,3-diaminophenazine (DAP) and carbon dots (CDs). Under the catalysis of horseradish peroxidase (HRP), the o-phthalylenediamine (OPD) was oxidized to DAP which had a yellow color and intense fluorescence. The color changes form colorless to yellow was used to design absorbance model immunoassay. Meanwhile, the absorption spectrum of DAP overlapped with the emission spectrum of CDs which caused the fluorescence of CDs to be quenched. The fluorescence changes of DAP and CDs were used to develop ratiometric fluorescence immunoassay. The dual-model immunoassay showed excellent sensitivity with the limits of detection (LODs) of 0.013 ng/mL for fluorescence mode and 0.062 ng/mL for absorbance mode. Meanwhile, both models exhibited great selectivity for AFB1. Additionally, the recovery rates suggested the proposed dual-model immunoassay had great potential in actual samples detection.

Texture Feature Extraction from 1H NMR Spectra for the Geographical Origin Traceability of Chinese Yam.

Adulteration is widespread in the herbal and food industry and seriously restricts traditional Chinese medicine development. Accurate identification of geo-authentic herbs ensures drug safety and effectiveness. In this study, 1H NMR combined intelligent "rotation-invariant uniform local binary pattern" identification was implemented for the geographical origin confirmation of geo-authentic Chinese yam (grown in Jiaozuo, Henan province) from Chinese yams grown in other locations. Our results showed that the texture feature of 1H NMR image extracted with rotation-invariant uniform local binary pattern for identification is far superior compared to the original NMR data. Furthermore, data preprocessing is necessary. Moreover, the model combining a feature extraction algorithm and support vector machine (SVM) classifier demonstrated good robustness. This approach is advantageous, as it is accurate, rapid, simple, and inexpensive. It is also suitable for the geographical origin traceability of other geographical indication agricultural products.

Robust online learning based on siamese network for ship tracking.

The complex and changeable inland river scenes resulting out of frequent occlusions of ships in the available tracking methods are not accurate enough to estimate the motion state of the target ship leading to object tracking drift or even loss. In view of this, an attempt is made to propose a robust online learning ship tracking algorithm based on the Siamese network and the region proposal network. Firstly, the algorithm combines the off-line Siamese network classification score and the online classifier score for discriminative learning, and establishes an occlusion determination mechanism according to the classification the fusion score. When the target is in the occlusion state, the target template is not updated, and the global search mechanism is activated to relocate the target, thereby avoiding object tracking drift. Secondly, an efficient adaptive online update strategy, UpdateNet, is introduced to improve the template degradation in the tracking process. Finally, on comparing the state-of-the-art tracking algorithms on the inland river ship datasets, the experimental results of the proposed algorithm show strong robustness in occlusion scenarios with an accuracy and success rate of 56.8% and 57.2% respectively. Supportive source codes for this research are publicly available at https://github.com/Libra-jing/SiamOL .

Breast cancer metastases to the thyroid and stomach: A case report.

The most common sites of metastasis for breast cancer are the soft tissues, bones, lungs, liver and brain; however, metastases to the gastrointestinal tract and thyroid gland from breast cancer rarely occur. The present study describes the case of a 30-year-old woman who developed gastric and thyroid metastases 5 years after her initial diagnosis of invasive ductal breast carcinoma. The initial pathological diagnosis when receiving modified radical mastectomy was invasive ductal carcinoma, and further immunohistochemical examination revealed the cancer to be estrogen receptor (-), progesterone receptor (-), human epidermal growth factor receptor 2 (HER2; ++) and Ki-67 (70%). Genetic testing indicated the HER2 amplification mutation, whereas BRCA1/2 testing was negative. A total of 21 months after surgery, during regular follow-up, the patient was revealed to have developed an enlarged lymph node in the left side of the neck and the first recurrence was confirmed. Approximately 5 years after surgery, the patient gradually developed multi-site metastasis, and developed metastases to the thyroid gland and stomach confirmed by pathology and imaging. Combined chemotherapy and targeted therapy were administered and exhibited good efficacy; however, the patient subsequently died due to heart failure. This case report describes the occurrence of gastric and thyroid metastases from breast cancer, and highlights the importance of distinguishing between metastatic and primary tumors. Distinguishing between a metastatic and primary tumor is crucial as treatment protocols vary significantly for these two types of tumors. For patients with a history of breast cancer it should first be considered whether they have metastasis of the primary disease or discomfort caused by treatment; however, the possibility of a second primary tumor cannot be ignored. If the patient has symptoms such as loss of appetite, nausea, vomiting, stomach pain and stomach discomfort, a gastroscopy should be performed in a timely manner.

A Survey of techniques for fine-grained web traffic identification and classification.

After decades of rapid development, the scale and complexity of modern networks have far exceed our expectations. In many conditions, traditional traffic identification methods cannot meet the demand of modern networks. Recently, fine-grained network traffic identification has been proved to be an effective solution for managing network resources. There is a massive increase in the use of fine-grained network traffic identification in the communications industry. In this article, we propose a comprehensive overview of fine-grained network traffic identification. Then, we conduct a detailed literature review on fine-grained network traffic identification from three perspectives: wired network, mobile network, and malware traffic identification. Finally, we also draw the conclusion on the challenges of fine-grained network traffic identification and future research prospects.

Hierarchical Harris hawks optimization for epileptic seizure classification.

The intelligent recognition of electroencephalogram (EEG) signals is a valuable tool for epileptic seizure classification. Given that visual inspection of EEG signals is time-consuming, and that mutant signals dramatically increase the workload of neurologists, automatic epilepsy diagnosis systems are extremely helpful. However, the existing epilepsy diagnosis methods suffer from some shortcomings. For example, they tend to fall into local optima quickly because of their failure to fully consider the discriminative features of EEG signals. To tackle this problem, in this article, an enhanced automatic epilepsy diagnosis method is proposed using time-frequency analysis and improved Harris hawks optimization (IHHO) with a hierarchical mechanism. Specifically, the signal is decomposed into five rhythms using continuous wavelet transform, with the local and global features extracted using the local binary pattern and the gray level co-occurrence matrix. Discriminative features are then selected and further mapped to the final recognition results using both IHHO and the k-nearest neighbor classifier. To evaluate its performance, the proposed method was compared with a variety of classical meta-heuristic algorithms on 23 benchmark functions. Moreover, the proposed approach achieved more than 99.67% accuracy on the Bonn dataset and 99.06% accuracy on the CHB-MIT dataset, out-performing a multitude of state-of-the-art methods. Taken together, these results demonstrate the utility of our approach in the automatic diagnosis of epilepsy. Supportive datasets and source codes for this research are publicly available at https://github.com/sstudying/lzzhen, and latest updates for the HHO algorithm are provided at https://aliasgharheidari.com/HHO.html.

Identification of significant genes and therapeutic agents for breast cancer by integrated genomics.

Breast cancer is the most commonly diagnosed malignancy in women; thus, more cancer prevention research is urgently needed. The aim of this study was to predict potential therapeutic agents for breast cancer and determine their molecular mechanisms using integrated bioinformatics. Summary data from a large genome-wide association study of breast cancer was derived from the UK Biobank. The gene expression profile of breast cancer was from the Oncomine database. We performed a network-wide association study and gene set enrichment analysis to identify the significant genes in breast cancer. Then, we performed Gene Ontology analysis using the STRING database and conducted Kyoto Encyclopedia of Genes and Genomes pathway analysis using Cytoscape software. We verified our results using the Gene Expression Profile Interactive Analysis, PROgeneV2, and Human Protein Atlas databases. Connectivity map analysis was used to identify small-molecule compounds that are potential therapeutic agents for breast cancer. We identified 10 significant genes in breast cancer based on the gene expression profile and genome-wide association study. A total of 65 small-molecule compounds were found to be potential therapeutic agents for breast cancer.

Deletion of the MBP1 Gene, Involved in the Cell Cycle, Affects Respiration and Pseudohyphal Differentiation in Saccharomyces cerevisiae.

Mbp1p is a component of MBF (MluI cell cycle box binding factor, Mbp1p-Swi6p) and is well known to regulate the G1-S transition of the cell cycle. However, few studies have provided clues regarding its role in fermentation. This work aimed to recognize the function of the MBP1 gene in ethanol fermentation in a wild-type industrial Saccharomyces cerevisiae strain. MBP1 deletion caused an obvious decrease in the final ethanol concentration under oxygen-limited (without agitation), but not under aerobic, conditions (130 rpm). Furthermore, the mbp1Δ strain showed 84% and 35% decreases in respiration intensity under aerobic and oxygen-limited conditions, respectively. These findings indicate that MBP1 plays an important role in responding to variations in oxygen content and is involved in the regulation of respiration and fermentation. Unexpectedly, mbp1Δ also showed pseudohyphal growth, in which cells elongated and remained connected in a multicellular arrangement on yeast extract-peptone-dextrose (YPD) plates. In addition, mbp1Δ showed an increase in cell volume, associated with a decrease in the fraction of budded cells. These results provide more detailed information about the function of MBP1 and suggest some clues to efficiently improve ethanol production by industrially engineered yeast strains. IMPORTANCE Saccharomyces cerevisiae is an especially favorable organism used for ethanol production. However, inhibitors and high osmolarity conferred by fermentation broth, and high concentrations of ethanol as fermentation runs to completion, affect cell growth and ethanol production. Therefore, yeast strains with high performance, such as rapid growth, high tolerance, and high ethanol productivity, are highly desirable. Great efforts have been made to improve their performance by evolutionary engineering, and industrial strains may be a better start than laboratory ones for industrial-scale ethanol production. The significance of our research is uncovering the function of MBP1 in ethanol fermentation in a wild-type industrial S. cerevisiae strain, which may provide clues to engineer better-performance yeast in producing ethanol. Furthermore, the results that lacking MBP1 caused pseudohyphal growth on YPD plates could shed light on the development of xylose-fermenting S. cerevisiae, as using xylose as the sole carbon source also caused pseudohyphal growth.

SPOP promotes CDCA5 degradation to regulate prostate cancer progression via the AKT pathway.

The E3 ubiquitin ligase adaptor Speckle-type POZ protein (SPOP) plays an important tumour suppressor role in prostate cancers (PCa), with mutation rate up to 15%. However, how SPOP mutations regulate prostate tumorigenesis remains elusive. Here, we report the identification of cell division cycle associated 5 (CDCA5) as a SPOP substrate. We found that SPOP interacts with CDCA5 and promotes its polyubiquitin degradation in a degron-dependent manner. This effect was greatly impaired by introducing PCa associated SPOP mutations. Importantly, we found that CDCA5 was essential for PCa cells to survive and proliferate. CDCA5 depletion in PCa cells led to cessation of proliferation, G2M arrest, severe sister chromatid aggregation disturbance, and apoptosis. we also found that CDCA5 knockdown decreased the protein expression of p-GSK3ß, increased the activity of caspase-3, caspase-9, and the Bax/Bcl-2 ratio. Besides, we confirmed that CDCA5 interrupted cancer cell behavior via the AKT pathway. In contrast, silencing SPOP or overexpressing CDCA5 increased cell proliferation. Consistently, depleting SPOP along with CDCA5, or overexpressing CDCA5 along with SPOP also caused the growth of cells repressed. Consistent with the functional role of CDCA5, the mRNA and protein levels of CDCA5 were significantly increased in PCa, compared to normal tissues, and its high expression was associated with more severe lymph node metastasis, higher Gleason score, and poorer prognosis. Together, our data showed that SPOP plays a crucial role in inhibiting tumorigenesis and partly achieved this by promoting the degradation of oncoprotein CDCA5.

Integrating Genome-Wide Association Studies and Gene Expression Profiles With Chemical-Genes Interaction Networks to Identify Chemicals Associated With Colorectal Cancer.

Colorectal cancer (CRC) is the third most common cancer and has the second highest mortality rate in global cancer. Exploring the associations between chemicals and CRC has great significance in prophylaxis and therapy of tumor diseases. This study aims to explore the relationships between CRC and environmental chemicals on genetic basis by bioinformatics analysis. The genome-wide association study (GWAS) datasets for CRC were obtained from the UK Biobank. The GWAS data for colon cancer (category C18) includes 2,581 individuals and 449,683 controls, while that of rectal cancer (category C20) includes 1,244 individuals and 451,020 controls. In addition, we derived CRC gene expression datasets from the NCBI-GEO (GSE106582). The chemicals related gene sets were acquired from the comparative toxicogenomics database (CTD). Transcriptome-wide association study (TWAS) analysis was applied to CRC GWAS summary data and calculated the expression association testing statistics by FUSION software. We performed chemicals related gene set enrichment analysis (GSEA) by integrating GWAS summary data, mRNA expression profiles of CRC and the CTD chemical-gene interaction networks to identify relationships between chemicals and genes of CRC. We observed several significant correlations between chemicals and CRC. Meanwhile, we also detected 5 common chemicals between colon and rectal cancer, including methylnitronitrosoguanidine, isoniazid, PD 0325901, sulindac sulfide, and importazole. Our study performed TWAS and GSEA analysis, linked prior knowledge to newly generated data and thereby helped identifying chemicals related to tumor genes, which provides new clues for revealing the associations between environmental chemicals and cancer.

Tamoxifen induces fatty liver disease in breast cancer through the MAPK8/FoxO pathway.

BACKGROUND: Prevention of metabolic complications of long-term adjuvant endocrine therapy in breast cancers remained a challenge. We aimed to investigate the molecular mechanism in the development of tamoxifen (TAM)-induced fatty liver in both estrogen receptor (ER)-positive and ER-negative breast cancer. METHODS AND RESULTS: First, the direct protein targets (DPTs) of TAM were identified using DrugBank5.1.7. We found that mitogen-activated protein kinase 8 (MAPK8) was one DPT of TAM. We identified significant genes in breast cancer and fatty liver disease (FLD) using the MalaCards human disease database. Next, we analyzed the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of those significant genes in breast cancer and FLD using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). We found that overlapping KEGG pathways in these two diseases were MAPK signaling pathway, Forkhead box O (FoxO) signaling pathway, HIF-1 signaling pathway, AGE-RAGE signaling pathway in diabetic complications, and PI3K-Akt signaling pathway. Furthermore, the KEGG Mapper showed that the MAPK signaling pathway was related to the FoxO signaling pathway. Finally, the functional relevance of breast cancer and TAM-induced FLD was validated by Western blot analysis. We verified that TAM may induce fatty liver in breast cancer through the MAPK8/FoxO signaling pathway. CONCLUSION: Bioinformatics analysis combined with conventional experiments may improve our understanding of the molecular mechanisms underlying side effects of cancer drugs, thereby making this method a new paradigm for guiding future studies on this issue.

Propranolol selectively inhibits cervical cancer cell growth by suppressing the cGMP/PKG pathway.

AIM: To observe the effect of propranolol in cervical cancer and investigate the mechanism of the effect． METHODS AND RESULTS: We found 5 direct protein targets (DPTs) of propranolol (PRO) by DrugBank5.0 firstly. Next, we analyzed protein-protein interaction (PPI) network and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of PRO DPTs and the result showed that PRO was linked with cGMP/PKG pathway. Then, we recognized the top 38 upexpressed genes of cervical cancer (CC) based original microarray datasets (GSE7803, GSE9750, GSE39001 and GSE63514). Further, we analyzed the biological process with the 38 overexpressed genes by STRING. We found some of overexpressed genes of CC participated in GMP biosynthetic process. Lastly, the function of PRO in CC was validated by MTT assay, Western blotting, flow cytometry and colony formation assay methods. We verified PRO can suppress cGMP/PKG pathway then inhibits CC cell growth. CONCLUSION: The bioinformatical analysis combine with traditional experiment can help us understanding potential molecular mechanism about how PRO acting in CC. This method is a new paradigm which can guide future researches about mechanism in existing diseases and drugs.

SPOP regulates the DNA damage response and lung adenocarcinoma cell response to radiation.

Speckle-type POZ protein (SPOP) plays an important role in maintaining genome stability. Disability or mutation of the SPOP gene has been reported to contribute to prostate cancer incidence and prognosis. However, the functions of SPOP in lung cancer remain poorly understood, especially in lung adenocarcinoma (LUAD). Here, we found that SPOP affects the LUAD cell response to radiation by regulating the DNA damage response (DDR) pathway. SPOP is widely expressed in lung cancer cell lines, and SPOP protein levels are upregulated when cells experience DNA damage. SPOP knockdown affects DDR repair kinetics, apoptosis and cell cycle checkpoints that are induced by IR (ionizing radiation). Furthermore, we found that SPOP positively regulates the expression of DDR factors Rad51 and Ku80. Taken together, these data indicate the essential roles of SPOP in the DDR signaling pathways and LUAD cell response to radiation.

Efficacy of Early Enteral Immunonutrition on Immune Function and Clinical Outcome for Postoperative Patients With Gastrointestinal Cancer.

BACKGROUND: Nutrition support is crucial for patients with gastrointestinal (GI) cancer after the operation. However, the controversy over the application of parenteral nutrition (PN) and early enteral immunonutrition (EEIN) has no determinate conclusion. MATERIALS AND METHODS: We compared the effects of PN and EEIN on the postoperative nutrition condition, immune status, inflammation level, long-term survival, and quality of life of the patients with GI cancer. Seventy-eight patients were randomly divided into the PN group (n = 44) or EEIN group (n = 34). After an 8-day nutrition treatment, clinical and immunological parameters were evaluated. RESULTS: The EEIN group had a significantly shorter hospital stay and higher body mass index level on postoperative day 30 than those in the PN group (P < .05). However, total hospital cost and incidences of short-term postoperative complications had no significant difference (P > .05). The percentages of CD4+ , natural killer, and natural killer T lymphocyte cells and the ratio of CD4+ /CD8+ in peripheral blood were significantly increased. Compared with the PN group, the EEIN group had a higher expression of activated cell surface markers such as CD27 and CD28. In addition, the secretion of interleukin (IL)-2 and interferon-γ was significantly higher, and the secretion of tumor necrosis factor-α and IL-10 was lower. Complication-free survival in the EEIN group were longer than those in the PN group (P = .04). CONCLUSION: EEIN is superior to PN in improving nutrition status, enhancing immune function, and elevating quality of life.

A Novel Acid-Stable Endo-Polygalacturonase from Penicillium oxalicum CZ1028: Purification, Characterization, and Application in the Beverage Industry.

Acidic endo-polygalacturonases are the major part of pectinase preparations and extensively applied in the clarification of fruits juice, vegetables extracts, and wines. However, most of the reported fungal endo-polygalacturonases are active and stable under narrow pH range and low temperatures. In this study, an acidic endo-polygalacturonase (EPG4) was purified and characterized from a mutant strain of Penicillium oxalicum. The N-terminal amino acid sequence of EPG4 (ATTCTFSGSNGAASASKSQT) was different from those of reported endopolygalacturonases. EPG4 displayed optimal pH and temperature at 5.0 and 60-70°C towards polygalacturonic acid (PGA), respectively, and was notably stable at pH 2.2-7.0. When tested against pectins, EPG4 showed enzyme activity over a broad acidic pH range (>15.0% activity at pH 2.2-6.0 towards citrus pectin; and >26.6% activity at pH 2.2-7.0 towards apple pectin). The Km and Vmax values were determined as 1.27 mg/ml and 5,504.6 U/mg, respectively. The enzyme hydrolyzed PGA in endo-manner, releasing oligo-galacturonates from PGA, as determined by TLC. Addition of EPG4 (3.6 U/ml) significantly reduced the viscosity (by 42.4%) and increased the light transmittance (by 29.5%) of the papaya pulp, and increased the recovery (by 24.4%) of the papaya extraction. All of these properties make the enzyme a potential application in the beverage industry.