Ochratoxin A (OTA) is a polyketide mycotoxin that commonly contaminates agricultural products and causes significant economic losses. In this study, the efficient OTA-degrading strain AP19 was isolated from vineyard soil and was identified as Acinetobacter pittii. Compared with growth in nutrient broth supplemented with OTA (OTA-NB), strain AP19 grew faster in nutrient broth (NB), but the ability of the resulting cell lysates to remove OTA was weaker. After cultivation in NB, the cell lysate of strain AP19 was able to remove 100% of 1 mg/L OTA within 18 h. The cell lysate fraction > 30 kDa degraded 100% of OTA within 12 h, while the fractions < 30 kDa were practically unable to degrade OTA. Further anion exchange chromatography of the > 30 kDa fraction yielded two peaks exhibiting significant OTA degradation activity. The degradation product was identified as OTα. Amino acid metabolism exhibited major transcriptional trends in the response of AP19 to OTA. The dacC gene encoding carboxypeptidase was identified as one of the contributors to OTA degradation. Soil samples inoculated with strain AP19 showed significant OTA degradation. These results provide significant insights into the discovery of novel functions in A. pittii, as well as its potential as an OTA decomposer.

The authors wish to make a change to the published paper [...].

Chitooligosaccharides (CHOS) have gained increasing attention because of their important biological activities. Enhancing the efficiency of CHOS production essentially requires screening of novel chitosanase with unique characteristics. Therefore, a rapid and efficient one-step affinity purification procedure plays important roles in screening native chitosanases. In this study, we report the design and synthesis of affinity resin for efficient purification of native chitosanases without any tags, using chitodisaccharides (CHDS) as an affinity ligand, to couple with Sepharose 6B via a spacer, cyanuric chloride. Based on the CHDS-modified affinity resin, a one-step affinity purification method was developed and optimized, and then applied to purify three typical glycoside hydrolase (GH) families: 46, 75, and 80 chitosanase. The three purified chitosanases were homogeneous with purities of greater than 95% and bioactivity recovery of more than 40%. Moreover, we also developed a rapid and efficient affinity purification procedure, in which tag-free chitosanase could be directly purified from supernatant of bacterial culture. The purified chitosanases samples using such a procedure had apparent homogeneity, with more than 90% purity and 10⁻50% yield. The novel purification methods established in this work can be applied to purify native chitosanases in various scales, such as laboratory and industrial scales.

•The LipY8p lipase from Yarrowia lipolytica was expressed in insect cells.•The lipase is a cold-active enzyme with the optimum temperature of 17 °C.•The lipase is stable over the pH ranges 5–9 with optimal activity at pH 7.5.•The lipase has a specific activity of 1102.9U/mg toward olive oil at 22 °C.•The effects of detergents, organic solvents on lipase activity were characterized.

沙雷氏蛋白酶属于锌金属蛋白酶M10B亚家族,存在于各种致病性革兰氏阴性细菌中.通常具有毒性,是一些疾病的关键致病因子.沙雷氏蛋白酶抑制剂是治疗这些疾病的有效药物,并且可以避免病原菌中的抗生素产生抗性.对沙雷氏蛋白酶和其抑制剂分别进行了综述,并简述了4种沙雷氏蛋白酶与抑制剂复合物相互作用的实例,以期为进一步了解沙雷氏蛋白酶的分泌调控机理和设计新型蛋白酶抑制剂药物提供参考.

Yarrowia lipolytica is a nonconventional model micro-organism with multiple biotechnological applications. It is also considered to be an excellent producer for lipase. Genome survey shows that Y. lipolytica possesses various paralogs of genes coding for extracellular, cell-bound, and intracellular lipolytic enzymes. However, little structural information on these isoenzymes is available. With the aim to facilitate crystal structure solution of Lip8, one of the most valuable lipases from Y. lipolytica, a less conventional protein expression techniqueselenomethionyl protein expression was used to produce recombinant selenomethionine (SeMet)-Lip8 in Escherichia coli. Finally, three Met residues of Lip8 were all substituted with SeMet. A total of 72mg of SeMet-Lip8 was obtained from a liter of the SeMet medium. Using sodium acetate as a precipitant and ammonium sulfate as an additive, crystals of the SeMet-Lip8 with 1.9 angstrom were successfully cultured through hanging-drop vapor diffusion method. The estimated crystal dimensions were 0.11x0.11x0.14mm(2). The crystal belonged to the space group I4 with unit cell parameters a=b=128.87 angstrom, c=171.77 angstrom, ===90 degrees. It is the second member of lipase crystal family from Y. lipolytica. This work will provide a platform for further studying lipases from a structural insight.

为获得较高的α-环糊精转化产率,采用单因素实验和响应面实验方法,筛选底物种类、底物浓度、加酶量、酶作用时间、作用温度和pH等多个单因素,对海洋芽孢杆菌Y112产的α-环糊精葡萄糖基转移酶产α-环糊精的条件进行了优化.然后利用Plackett-Burman实验筛选得到影响α-环糊精转化率的3个主要因素:底物浓度、温度和pH值.最终采用响应面分析法得到的最佳转化条件为马铃薯淀粉浓度为5％,加酶量200 U/g(淀粉),pH值为8.4,温度30℃,200 r/min反应6h,α-环糊精转化率均值为28.67％,比优化前的产率提高了2.48倍.

Alginate, the most abundant carbohydrate presents in brown macroalgae, has recently gained increasing attention as an alternative biomass for the production of biofuel. Oligoalginate lyases catalyze the degradation of alginate oligomers into monomers, a prerequisite for bioethanol production. In this study, two new oligoalginate lyase genes, oalC6 and oalC17, were cloned from Cellulophaga sp. SY116, and expressed them in Escherichia coli. The deduced oligoalginate lyases, OalC6 and OalC17, belonged to the polysaccharide lyase (PL) family 6 and 17, respectively. Both showed less than 50% amino acid identity with all of the characterized oligoalginate lyases. Moreover, OalC6 and OalC17 could degrade both alginate polymers and oligomers into monomers in an exolytic mode. Substrate specificity studies demonstrated that OalC6 preferred α-L-guluronate (polyG) blocks, while OalC17 preferred poly β-D-mannuronate (polyM) blocks. The combination of OalC6 and OalC17 showed synergistic degradation ability toward both alginate polymers and oligomers. Finally, an efficient process for the production of alginate monomers was established by combining the new-isolated exotype alginate lyases (i.e., OalC6 and OalC17) and the endotype alginate lyase AlySY08. Overall, our work provides new insights for the development of novel biotechnologies for biofuel production from seaweed.

The development of antitumor drugs has attracted cancer researchers and the identification of novel antitumor lead compounds is certainly of great interest. The fermentation broth of Bacillus sp. N11-8, which was isolated from the Antarctic waters, showed cytotoxicity towards different cells. A cytotoxic polypeptide, PBN11-8, was purified from the fermentation broth of Bacillus sp. N11-8 using ultrafiltration, ammonium sulfate precipitation, anion exchange liquid chromatography and high performance liquid chromatography (HPLC). Cloning and sequence analysis showed that PBN11-8 polypeptide (MW: ~19 kDa by the electrospray-ionization (ESI)) displayed high similarity with peptidase M84 from Bacillus pumilus. PBN11-8 possessed moderate cytotoxicity towards several cancer cell lines with IC50 values of 1.56, 1.80, 1.57, and 1.73 µg/mL against human hepatocellular carcinoma cell line BEL-7402, human renal clear cell adenocarcinoma cell line 786-0, human hepatocellular carcinoma cell line HepG2, and human pancreatic cancer cell line Panc-28, respectively. Moreover, the polypeptide displayed weak cytotoxicity towards normal cell line renal tubular epithelial cell line HK2 and human normal liver cell line L02 cells. Wound healing migration and Transwell experiments demonstrate that PBN11-8 could inhibit the migration and invasion of BEL-7402. Further investigation revealed that PBN11-8 suppresses focal adhesion kinase (FAK)-mediated adhesion, migration, and invasion by disturbing FAK/extracellular regulated protein kinases (ERK) signaling and matrix metalloproteinase-2(MMP-2) and matrix metalloproteinase-9 (MMP-9) in BEL-7402 cells. Thus, PBN11-8 represents a potential novel anti-cancer lead compound.

采用6种不同孔径和亲疏水性的聚苯乙烯微球,通过吸附法固定化脂肪酶YS2071,筛选得到最佳载体,对固定化条件进行了优化并对其性能进行了研究.结果表明:粒径范围0.6～1 μm的氨基聚苯乙烯微球B具有最佳的固定化效果,在酶质量浓度5 mg/mL、pH 10、固定化温度20℃、振荡转速250 r/min、吸附时间6h条件下,酶活回收率达到42.82％;与YS2071脂肪酶游离酶相比,经固定化后的载体脂肪酶的温度稳定性、金属离子稳定性、有机溶剂稳定性、pH稳定性均明显增强;25℃存储30 d后,固定化酶酶活保留率比游离酶酶活保留率提高了15.09个百分点;固定化酶重复使用5次后,酶活保留率为63.77％.

Objective:To establish an efficient method for biomimetic affinity purification of marine metalloprotease MP.Methods:Using the biomimetic affinity materials to optimize the purification conditions by response surface optimization method,such as the single factor experiment,Plackett-Burman experiment design,Box-Behnken experiment.And the purity of purified samples by SDS-PAGE and HPLC was analyzed.Results:The purification conditions were optimized by single factor experiment and the response surface design.The study indicated that the best purification conditions of the boronic acid gel were:When enzyme concentration was 100 mg/mL,the optimal loading buffer was glycine-sodium hydroxide of pH8.6 and the sample rate was 1 mL/min.The elution buffer was disodium hydrogen phosphate-citric acid of pH5.2,and elution rate was 2 mL/min,the elution buffer added to mannitol of 75 mmol/L,the purification efficiency was enhanced 11 times and the purity above 98.8％.

Cyclodextrin glycosyltransferase (CGTase) is an enzyme able to convert starch and other substrates into cyclodextrins (CDs). A marine strain Y112 producing α-CGTase was identified as Bacillus agaradhaerens Y112 by physiological and biochemical characterization, and 16S rDNA analysis. The gene coding for α-CGTase was cloned, sequenced and expressed in Escherichia coli BL21 (DE3) cells. Recombinant α-CGTase was purified in one-step chromatographic separation and its purity evaluated by SDS-PAGE, showing the presence of one band with a molecular mass of about 92 kDa. Additionally, enzymatic capability was analyzed by measuring the starch conversion, and resulted in about 45% of CDs obtained after 6 h of cyclodextrin reaction. Of these CDs, mainly α-CD was produced (70% of the total CDs yield), suggesting the potential of this CGTase for industrial applications.

Food microbiology has been revolutionized by the advances in molecular genetic technology of microbial ecology.Novel microbial detection methods have been applied in the study of processing and storage of aquatic food products.The microbial diversity in the sample to be studied could be detected by high-throughput sequencing (HTS) approaches as culture-independent analyses.The aim of this review is to give a brief introduction of the microbial amplicon HTS technology,to present a survey of recent aquatic food investigations via 16S rRNA amplicon HTS and to illustrate the potential of this approach for a valuable tool in the better characterization of aquatic foods and their processing and storage.

The effect of different resins on immobilizing lipase ADM47601 was investigated in this study. A total of 21 types of resins were screened, and the anion-exchange resin D316 demonstrated the best immobilization. After the orthogonal test, the recovery ratio of enzyme activity reached up to 65.53% ± 1.06% under optimal conditions: 10000U lipase per 1g resin, pH7.5, 25℃, 150r/min and 3 h for immobilization. The optimal pH of immobilized lipase was 7.5, and it exhibited 85% of the maximum activity in the range of pH6.0–9.0. The optimal temperature was 40℃, and the activity was maintained at 66% after 1 h of storage at 50℃. At 25℃, the half-life was 55 days and the residual activity was still higher than 60% after utilizing the lipase 8 times. The immobilized lipase can maintain activity in various organic reagents, and its Km and Vmax values were 4.86 × 10?5mol/L and 1.31 × 10?5mol/(L·min), respectively, indicating high affinity to the substrate.

Free lipase is difficult to achieve continuous operation.In order to improve the efficiency of lipase and apply it to the industrialized production,the technology of immobilized lipase YS2071 was studied by screening the optimal carrier and using single factor experiment method to optimize lipase immobilized conditions.Using MI-BSI resin as carrier and genipin as cross-linking agent,adsorption-crosslinking method was employed to maintain relative high enzyme activity.Immobilized conditions were studied by using PB experiment and the response surface design.The optimal conditions of immobilized lipase YS2071 were as followes:the enzyme dosage was 8 mg,the adsorption time was 8 h,the adsorption temperature was 20 ℃,the initial pH was 8.6,the concentration of genipin was 0.48 g/L,the crosslinking time was 1 h.Under these conditions,the recovery rate of immobilized enzyme was up to 60％.

The alkaline metalloproteases MP is extracted from the strain YS-80-122 and belongs to the family of serralysin.Similarly to the other enzymes of this family reported before,there is an inhibitor gene lupI downstream of the MP gene and LupI can completely inhibit the activity of MP.On the basis of the wild-type inhibitor gene,four amino acids were added to the N-terminus of LupI,named LupI-MSSS.This work aims to construct a prokaryotic expression vector for LupI-MSSS,the recombinant plasmid was transformed to BL21 (DE3),and the expression was induced by IPTG.The recombination LupI-MSSS was separated using SDS-PAGE and the size of expressed LupI-MSSS was consistent with the prediction.Single factor experiment were used to optimize the fermentation condition and the optimal conditions were:the inoculation amount is 2 ％,IPTG as the induced agent was added at 3 h after inoculating,and it~ final concentration is 0.5 mmol/L,the recombined E.coli need inducing 8 hours at 37 ℃.Consecutive steps were used to achieve the purified protein as follows:ultrafiltration,Q Sepharose ion exchange,Superdex 200 gel filtration,and the purity of LupI-MSSS is up to 99％.

In order to study microbial diversity in the surface seawater of the Drake Passage in Antarctic,7 surface seawater samples were collected to isolate the strains of culturable yeast by the traditional method,and 18S rDNA sequencing and phylogenetic analyses were carried out.The results showed that 45 yeast strains were isolated and there were 9 genera and 12 species.Meyerozyma sp.and Meyerozyma guilliermondii was the dominant genera and species.The results of the catalase activities showed that 23 strains were strong catalase positive,13 strains were weak,and 80％ strains were catalase positive.

ι-Carrageenases play a role in marine ι-carrageenan degradation, and their enzymatic hydrolysates are thought to be excellent antioxidants. In this study, we identified a new ι-carrageenase, encoded by cgiF, in psychrophilic bacterium Flavobacterium sp. YS-80-122. The deduced ι-carrageenase, CgiF, belongs to glycoside hydrolase family 82 and shows less than 40% amino acid identity with characterized ι-carrageenases. The activity of recombinant CgiF peaked at 30°C (1,207.8U/mg). Notably, CgiF is a cold-adapted ι-carrageenase, which showed 36.5% and 57% of the maximum activity at 10°C and 15°C, respectively. In addition, it is a thermo-tolerant enzyme that recovered 58.2% of its initial activity after heat shock. Furthermore, although the activity of CgiF was enhanced by NaCl, the enzyme is active in absence of NaCl. This study also shows that CgiF is an endo-type ι-carrageenase that hydrolyzes β-1,4-linkages of ι-carrageenan, yielding neo-ι-carratetraose as the main product. Its cold-adaptation, thermo-tolerance, NaCl independence and high neo-ι-carratetraose yield make CgiF an excellent candidate for industrial applications in production of ι-carrageen oligosaccharides from seaweed polysaccharides.

The metalloproteinase MP belongs to the serralysin family, which is involved in important functions such as nutrient acquisition and infection pathogenesis. Serralysin proteases in highly purified form are commonly used at the industrial level with several purposes. In this study, we set up an efficient and rapid purification protocol for MP using a p-aminobenzamidine-modified affinity chromatography. The affinity medium was synthesized by using p-aminobenzamidine as affinity ligand immobilized via cyanuric chloride spacer to Sepharose 6B sorbent carrier. According to the adsorption analysis, the dissociation constant Kd and theoretical maximum adsorption Qmax of this medium were 24.2 μg/mL and 24.1 mg/g wet sorbent, respectively. The purity of MP was assessed by a high-performance liquid chromatography on a TSK3000SW column and sodium dodecyl sulfate polyacrylamide gel electrophoresis, revealing values of 98.7 and ∼98%, respectively. The specific activity of purified MP was 95.6 U/mg, which is similar to values obtained through traditional purification protocols. In conclusion, our protocol could be easily employed for the rapid isolation of MP with high purity, and could be implemented for other serralysin family proteases.

Unsaturated alginate disaccharides (UADs), enzymatically derived from the degradation of alginate polymers, are considered powerful antioxidants. In this study, a new high UAD-producing alginate lyase, AlySY08, has been purified from the marine bacterium Vibrio sp. SY08. AlySY08, with a molecular weight of about 33 kDa and a specific activity of 1070.2 U/mg, showed the highest activity at 40 °C in phosphate buffer at pH 7.6. The enzyme was stable over a broad pH range (6.0–9.0) and retained about 75% activity after incubation at 40 °C for 2 h. Moreover, the enzyme was active in the absence of salt ions and its activity was enhanced by the addition of NaCl and KCl. AlySY08 resulted in an endo-type alginate lyase that degrades both polyM and polyG blocks, yielding UADs as the main product (81.4% of total products). All these features made AlySY08 a promising candidate for industrial applications in the production of antioxidants from alginate polysaccharides.