Enhanced cold active lipase production by metagenomic library recombinant clone CALIP3 with a step-wise temperature and dissolved oxygen level control strategy

A metagenomic library recombinant clone CAPL3, an Escherichia coli strain generated by transformed with metagenomic library from deep-sea sediments, can efficiently produce cold active lipase. The effects of both temperature and dissolved oxygen（DO） on cold active lipase production by batch culture of metagenomic library recombinant clone（CAPL3） from deep-sea sediment were investigated. First, a two-stage temperature control strategy was developed, in which the temperature was kept at 34 ℃ for the first 15 h, and then switched to30 ℃. The cold active lipase activity and productivity reached 315.2 U·ml^-1and 8.08 U·ml^-1·h^-1, respectively,increased by both 14.5% compared to the results obtained with temperature controlled at 30℃. In addition, different DO control modes were conducted, based on the data obtained from the different DO control strategies and analysis of kinetics parameters at different DO levels. A step-wise temperature and DO control strategy were developed to improve lipase production, i.e., temperature and DO level were controlled at 34℃, 30% during 0–15 h;30 ℃, 30% during 15–18 h, and 30 ℃, 20% during 18–39 h. With this strategy, the maximum lipase activity reached 354.6 U·ml^-1at 39 h, which was 28.8% higher than that achieved without temperature and DO control（275.3 U·ml^-1）.

Lipase inhibitory activity of Lagenaria siceraria fruit as a strategy to treat obesity

Objective:To explore pancreatic lipase inhibitory activity under different extraction conditions in order to track the most potent extract.Methods:The methanolic extract and its fractions in solvents of increasing polarity,ether,chloroform,ethyl acetate,n-butanol and water,were made through cold maceration.Extracts in ethanol,ethyl acetate,acetone and chloroform were similarly prepared.Aqueous extract was prepared through hot decoction method.A reported method was used to determine lipase inhibitory activity of extracts and fractions over wide ranges of concentrations.Results:The extracts and fractions exhibited concentration dependent activity.The IC50(μg/mL)values of methanolic,ethanolic,chloroform,ethyl acetate,acetone,ethyl acetate(after washing with water)and aqueous decoction were 293.40,266.47,157.59,182.12,352.34,257.00,and 190.00,respectively.The activity of chloroform,ethyl acetate and aqueous extracts were close to that of the drug orlistat(IC50 146μg/mL).Out of the fractions of the methanolic extract,the chloroform fraction was most active(IC50 189.6μg/mL).The order of inhibitory activity of the fractions was as follows:chloroform>ether>n-butanolic>aqueous>ethyl acetate.The GC/MS analysis of the most active chloroform faction showed the presence of hexadecanoic acid,methyl hexadecanoate,isopropyl palmitate,methyl 9,12-octadecadienate,and methyl 9,12,15-octadecatrienoate.Conclusions:The study suggests that Lagenaria siceraria has potential to inhibit pancreatic lipase activity,suppressing lipid digestion and thereby diminishing entry of lipids into the body.Regular intake of aqueous decoction of the fruit may therefore be recommended for control of obesity.Fatty acids and their esters may play role as inhibitors of lipase.

Conjugation of a zwitterionic polymer with dimethyl chains to lipase significantly increases the enzyme activity and stability

Enzyme-polymer conjugates are complex molecules with great practical significance.This work was designed to develop a novel enzyme-polymer conjugate by covalently coupling a zwitterionic polymer with side dimethyl chains(pID)to Candida rugosa lipase(CRL)via the reaction between the anhydrides of polymer chains with the amino groups of the enzyme.The resulting two CRL-pID conjugates with different pID grafting densities were investigated in term of the catalytic activity,stability and structural changes.In comparison with native CRL,both the CRL conjugates displayed 2.2 times higher activity than the native enzyme,and showed an increase in the maximum reaction rate(V_(max))and a decrease in the Michaelis constant(K_(m)),thus resulting in about three-fold increases in the catalytic efficiency(k_(cat)/K_(m)).These are mainly attributed to the activation of lipase by the hydrophobic alky side chains.Moreover,the thermostability and pH tolerance of the lipase conjugates were significantly enhanced due to the stabilizing effect of the zwitterion moieties.For instance,a five-fold increase of the enzyme half-life at 50℃ for the high-pID conjugated CRL was observed.Spectroscopic studies reveal that the pID conjugation protected the enzyme in the changes in its microenvironment and conformation,well correlating with enhanced activity and stability of lipase conjugates.The findings indicate that enzyme conjugation to the zwitterionic polymer is promising for improving enzyme performance and deserves further development.

The effects of steaming and roasting treatments on lipase activity and nutritional components of “oat rice” (OR): the peeled naked oat (Avena nuda) kernels

Peeled naked oat kernels, named “oat rice” (OR) by Chinese food scientists and processors, are novel oat products in China. This study exam-ined the effects of steaming and roasting treat-ments on the enzyme activities, nutritional con-tents, and flour pasting properties of OR kernels. Results showed that a peeling time of 20 s caused 16.13% β-glucan loss, while a peeling time 25 s caused 34.29% β-glucan loss in the kernels. OR kernels with a 20 s peeling treatment demonstrated significantly higher starch levels and kernel whiteness compared with normal oat kernels (P<0.01). It was also found that normal pressure steaming, autoclaved steaming and infrared roasting treatments could exterminate lipase activities in the OR kernels, and provide the OR kernels with significantly lower final viscosities and setback values than normal kernels (P<0.01).

Fermentation Performance and Characterization of Cold-Adapted Lipase Produced with Pseudomonas Lip35

Strain of Pseudomonas Lip35 producing lipase was isolated in a refrigerator. Lipase production and characterization of this strain were investigated under different conditions. The Pseudomonas was cultivated in shaking flasks in a fermentation medium in various nutritional and physical environments. Lipase production has been influenced by the presence of yeast-extract, soybean powder, NaCI, and Tween-80. Maximum lipase productivity was obtained when the physical environment of the fermentation medium was optimal for 67 h. The production of lipase reached 58.9 U·mL^-1. The lipase of Pseudomonas Lip35 can be considered to be inducible, but the inducer had little influence on the production of lipase. The lipase was characterized and showed high lipolytic activity from pH 7.5-8.0. The optimum temperature was observed at 20℃ and the thermal inactivation of lipase was obvious at 60℃. The lipase activity was inhibited by K＋, stimulated by Ca^2＋, and thermostability decreased in the presence of Ca^2＋, therefore the lipase was Ca^2＋ -dependent cold-adapted enzyme.

Isolation and characterization of glacier VMY22, a novel lytic cold-active bacteriophage of Bacillus cereus

As a unique ecological system with low temperature and low nutrient levels, glaciers are considered a "living fossil" for the research of evolution. In this work, a lytic cold-active bacteriophage designated VMY22 against Bacillus cereus MYB41-22 was isolated from Mingyong Glacier in China, and its characteristics were studied. Electron microscopy revealed that VMY22 has an icosahedral head(59.2 nm in length, 31.9 nm in width) and a tail(43.2 nm in length). Bacteriophage VMY22 was classified as a Podoviridae with an approximate genome size of 18 to 20 kb. A one-step growth curve revealed that the latent and the burst periods were 70 and 70 min, respectively, with an average burst size of 78 bacteriophage particles per infected cell. The pH and thermal stability of bacteriophage VMY22 were also investigated. The maximum stability of the bacteriophage was observed to be at pH 8.0 and it was comparatively stable at p H 5.0–9.0. As VMY22 is a cold-active bacteriophage with low production temperature, its characterization and the relationship between MYB41-22 and Bacillus cereus bacteriophage deserve further study.

基于AH理念的寒冷地区工业厂房改造设计研究——以2022年Active House Award中国区竞赛获奖方案为例

在“碳中和、碳达标”的时代目标下,工业厂房改造作为城市更新的一环,绿色低碳应作为其改造原则予以重视。总结2022年第三届AHA中国区竞赛获奖作品“穿廊叠拱,绿厂新生——北京市大兴区天普东厂区改建方案设计”的设计构思,针对北京夏热冬冷的特定气候,方案以主动式建筑理念为出发点,在设计层面对其功能和空间更新解析后,从适度合理的主动性、以人为本的舒适性、高效率的能源手段和可持续的环境措施四个角度,对建筑的弹性设计、引导用户行为、自然采光与通风、太阳能多维利用、建筑的气候适应性策略等方面进行重点分析,为我国寒冷地区的工业厂房的绿色低碳改造设计提供借鉴。

Steeping of Whole Dried Maize Seeds in Buffers Altered Fatty Acid Ionization State, Composition, and Lipase Activity

Steeping is a simple model of studying the activation and modulation of the physiological pathways involved in seed germination. In this study, steeping of grains of the ‘obatanpa’ maize variety in buffers at different pH was monitored through the measurements of lipase activity, oil yield, fatty acid component and unsaturation, and germination capacity. Lipase activity of grains steeped for four days decreased in the order: pH 3 > pH 5 > pH 7 > pH 9 > pH 11. Decreasing lipase activity was corroborated with decreasing free fatty acid components, protein concentrations and oil yields. The unsaturation components of the oil fractions only marginally increased with increasing steeping media pH. Three major components were detected by TLC in all oil fractions. The unique components were confirmed by their uniform UV-absorption spectra converging at an isosbestic point of 290 nm. Germination capacity was much reduced for seeds steeped in buffered media for 24 hours compared with seeds steeped in portable water though the pattern of germination, which was monitored for five days, did not change. This study has demonstrated the use of pH changes of steeping medium to modulate physicochemical properties and germination of seeds. The physicochemical changes were observed after seeds have been submerged under steeping buffer for four days. Practical application: With proliferation of specialty maize hybrids, the study provides an insight into the development of experimental protocols for the selection of types of maize grain for preparation of foods and beverages in terms of general characterization and lipolytic activity, which have implications for flavor, taste and odor of the final products. The imminence of this in some traditional ways of preparing malted and fermented maize foods and beverages, which go through days of steeping, cannot be overemphasized. This study therefore provides another dimension to the manipulation of the steeping stage to develop varieties of maize-based product.

Application of a nondestructive method to evaluate the active layer in a cold region

To provide a safe transportation system in an extremely cold region,evaluation needs to be conducted of the thickness and the volumetric water content of the active layer,as they significantly affect frost heave.The objective of this study was to evaluate the dielectric constant(κ)of the active layer using ground-penetrating radar(GPR)and a dynamic cone penetrometer(DCP);this evaluation was then used to estimate the thickness and the volumetric water content of the active layer.A field located in midwest Alaska was selected as the study site.A GPR survey and two DCP tests were conducted on the surface of the ground,and the ground temperature was measured.From the GPR survey,travel times of the electromagnetic wave in the active layer were obtained.In addition,the thickness of the active layer was determined by using the dynamic cone penetration index(DCPI)and ground temperature.By using the travel time and travel distance of the electromagnetic wave in the active layer,dielectric constants were calculated as 26.3 and 26.4 for two DCP points.From the mean dielectric constant,the volumetric water content was estimated to be 40%~43%,and the thickness of the active layer was evaluated along the GPR survey line.The spatial-scaled GPR image showed that the thickness of the active layer varied from 520 mm to 700 mm due to the presence of a puddle,which accelerated the heat exchange.The results show that evaluation of the dielectric constant using the GPR survey and the DCP test can be effectively used to estimate the thickness and the volumetric water content of the active layer.

Cloning and heterologous expression of pro-2127,a gene encoding cold-active protease from Pseudoalteromonas sp.QI-1

The psychrotropic bacterium, Pseudoalteromonas sp. QI-I, which produces extracellular cold-active protease, was isolated from Antarctic seawater. The genomic DNA of this bacterium was used to construct a plasmid genomic library with the goal of screening cold-active protease genes. Gene pro-2127 with an open reading frame of 2127 bp encoding protease PRO-2127 was cloned and sequenced. Alignment of amino acid sequences suggested that the precursor of PRO-2127 was a member of subfamily S8A, and that it might contain four domains： a signal peptide, an N-terminal prosequence, a catalytic domain and a C-terminal extension. Amino acids Asp185, His244 and Ser425 might form a catalytic triad. PRO-2127 showed some structural features common to psychrophilic enzymes, such as a decrease in Arg residues and the Arg/（Arg＋Lys） ratio. Heterologous expression of pro-2127 in Escherichia coli BL21 （DE3） by pColdlII was also successfully observed in this study.

Purification and Properties of Cold-active Metalloprotease from Curtobacterium luteum and Effect of Culture Conditions on Production

Curtobacterium luteum, a gram-positive psychrotrophic bacterium, secreting an extracellular protease was isolated from the soil of Gangotri glacier, Western Himalaya. The maximum enzyme production was achieved when isolate was grown in a pH-neutral medium containing skim milk at 15oC over 120 hour. The metal ions such as Zn2+ and Cr2+ enhanced enzyme production. The specific activity of purified enzyme was 8090 u/mg after 34.1 fold purification. The 115 kD enzyme was a metalloprotease (activity inhibited by EDTA and EGTA) and showed maximum activity at 20oC and pH 7. The enzyme was active over a broad pH range and retained 84% of its original activity between pH 6–8. There was no loss in enzyme activity when exposed for 3 hours at 4oC-20oC. However, lost 65% of activity at 30oC, and was almost inactivated at 50oC, but was resistant to repeated freezing and thawing. The enzyme activity was stimulated by manganese ions; however, it was inactivated by copper ions.

Purification and Characterization of Cold-active α-Amylase Excreted by A Strain of Marine Cold-adaptive Penicillia

The filamentous fungi from the Huanghai sea sludge were screened according to their ability to produce cold-active α-amylase. The strain with the highest amylase activity was identified as Penicillium species. The α-amylase purified by ammonium sulphate precipitation and column chromatography on DEAE-sepharose and sephadex G-100 shows a molecular weight of about 55000 and a pI of 4.38. The enzyme is stable in a pH range of 5.5—8.0 and has a maximum activity at pH 6.0. Compared with the α-amylase from mesophiles and thermophiles, the cold-active enzyme shows a high enzyme activity at lower temperatures and a high sensitivity at temperatures higher than 50 ℃. The optimal temperature is 40 ℃ and the activity decreases dramatically at temperatures above 50 ℃. Ca 2+ shows a significant effect on maintaining the structure and the activity of the enzyme. EDTA and Cu 2+ are its inhibitors. The products from the hydrolysis of soluble starch with the cold-active enzyme are maltose and other oligosaccharides.

Study on the Determination Conditions of Lipase Activity and the Effects of Commercial Detergent Products

The activity of lipase was determined by alkaline titration method and the conditions for the determination of lipase activity were studied. The results showed that the lipase activity was stable when the volume fraction of olive oil was 25% and the temperature was at 40℃ with PH=7.5. Under the proper conditions, the effects of anionic and nonionic surfactants were investigated. The effect of nonionic surfactants on the activity of lipase was less than that of anionic surfactants. Finally, the analysis method was used to determine the activity of the lipase in the commercial detergent products. The results showed that the method can be used for the determination of lipase activity in both liquid detergent and laundry powder. However, the temperature and pH value of the detergent solution should be controlled strictly during the conduction of the determination.

Molecular Cloning and Characterization of a New Cold-active Extradiol Dioxygenase from a Metagenomic Library Derived from Polychlorinated Biphenyl-contaminated Soil

To find new extradiol dioxygenases（EDOs,EC 1.13.11.2）,a metagenomics library was constructed from polychlorinated biphenyl-contaminated soil and was screened for some dioxygenase with aromatic ring cleavage activity.A novel EDO,designated as BphC_A,was identified and heterologously expressed in Escherichia coli.The deduced amino acid sequence of BphC_A exhibited a homology of less than 60% with other known EDOs.Phylogenetic analysis of BphC_A suggests that the protein is a novel member of the EDO family.The enzyme exhibits higher substrate affinity and catalytic efficiency toward 3-methylcatechol than toward 2,3-dihydroxybiphenyl or catechol,the preferred substrate of other known EDOs.The optimum activity of purified BphC_A occurred at pH=8.5 and 35 °C,and BphC_A showed more than 40% of its initial activity at 5 °C.The activity of purified BphC_A was significantly induced by Mn^2＋ and slightly reduced by Al^3＋,Cu^2＋ and Zn^2＋.

器壁固定化脂肪酶(Pseudomonas cepacia lipase)的非水活性与稳定性

许多脂肪酶在有机体系中表现出催化作用,可用于绿色有机合成.但其催化活性和稳定性明显低于水/油(有机相)界面上的表现.为了提高脂肪酶在有机反应体系中的活性和稳定性,依据脂肪酶的界面活化机制,以水为酶蛋白构象优化剂、羧甲基纤维素为赋形剂,通过物理吸附的方式,将典型的假单胞菌脂肪酶(Pseudomonas cepacia lipase)固定在锥形瓶的内壁上,形成简易的生物反应器.为方便检测器壁固定化酶促反应动力学,选择特征吸收为640 nm的生化指示剂2,6-二氯靛酚为反应底物,乙酸乙烯酯为酰化试剂,丙酮为溶剂.光谱检测表明,催化反应0.5 h后,器壁固定化脂肪酶转化底物的能力是脂肪酶粉的6倍.在每次催化5 h共10次的循环催化中,器壁固定化脂肪酶的催化活性平均每次仅降低3.2%,而酶粉降低11.8%.结果表明,该器壁固定化脂肪酶的活性和稳定性相对于酶粉明显提高,这将为通过固定化有效提高脂肪酶的非水催化作用提供重要的参考.

基于分子动力学的脂肪酶Lipase 5的热稳定性研究

天然的低温脂肪酶往往结构热稳定性比较差,制约了其长时间有效地发挥催化作用及保存.该研究以来源于白色念珠菌(Candida albicans)的低温脂肪酶Lipase 5为对象,运用相关分子动力学方法进行研究,提出了提高其热稳定性的理论策略.首先运用同源建模方法构建目标蛋白的三维结构模型;然后通过18ns分子动力学模拟,锚定目标蛋白不稳定区域中柔性氨基酸(甘氨酸)的位置,并将这些柔性氨基酸位点突变为刚性氨基酸(脯氨酸);最后利用分子动力学模拟来验证这些突变对蛋白质热稳定性的影响.结果发现,将Lipase 5三维结构中的第279位甘氨酸突变为脯氨酸后,使得蛋白质热稳定性增强.这为类似低温脂肪酶的热稳定性改造的实验设计提供了理论支持.

Zwitterionic polymer-coated porous poly(vinyl acetate–divinyl benzene)microsphere: A new support for enhanced performance of immobilized lipase

Enzyme immobilization has attracted great attention for improving the performance of enzymes in industrial applications.This work was designed to create a new support for Candida rugosa lipase(CRL)immobilization.A porous poly(vinyl acetate–divinyl benzene)microsphere coated by a zwitterionic polymer,poly(maleic anhydride-alt-1-octadecene)and N,N-dimethylethylenediamine derivative,was developed for CRL immobilization via hydrophobic binding.The catalytic activity,reaction kinetics,stabilities and reusability of the immobilized CRL were investigated.It demonstrated the success of the zwitterionic polymer coating and subsequent CRL immobilization on the porous microsphere.The immobilized lipase(p2-MS-CRL)reached27.6 mg·g^-1 dry carrier and displayed a specific activity 1.5 times higher than free CRL.The increase of Vmax and decrease of Kmwere also observed,indicating the improvement of catalytic activity and enzyme-substrate affinity of the immobilized lipase.Besides,p2-MS-CRL exhibited significantly enhanced thermal stability and pH tolerance.The improved performance was considered due to the interfacial activation regulated by the hydrophobic interaction and stabilization effect arisen by the zwitterionic polymer coating.This study has thus proved the advantages of the zwitterionic polymer-coated porous carrier for lipase immobilization and its potential for further development in various enzyme immobilizations.

Fabrication and characterization of epoxylated zwitterionic copolymergrafted silica nanoparticle as a new support for lipase immobilization

Our previous work proved that the thermal stability of Candida rugosa lipase(CRL)immobilized on zwitterionic polymer(poly(carboxybetaine methacrylate))grafted silica nanoparticle(SNP)was much higher than that on poly(glycidyl methecrylate)(pGMA)grafted SNP,while the latter showed significantly increased activity.Inspired by the research,we have herein proposed to synthesize copolymers of zwitterionic sulfobetaine methacrylate(SBMA)and GMA for CRL immobilization.The copolymers were grafted onto SNP surface at three GMA/SBMA(G/S)molar ratios(G100/S0,G50/S50,G10/S90),followed by the covalent coupling of CRL to the surface copolymers.The immobilized CRLs on the corresponding supports were denoted as p(G100-S0)-CRL,p(G50-S50)-CRL and p(G10-S90)-CRL.The enzyme loading increased with the increase of GMA content in the copolymer,while the activity varied with the grafted copolymer composition.Kinetic study proved the improvement of enzyme-substrate affinity after immobilization.In comparison to p(G100-S0)-CRL,p(G50-S50)-CRL and p(G10-S90)-CRL presented remarkably enhanced thermal stability and pH tolerance,and p(G10-S90)-CRL showed the highest stability.These results suggest that the copolymer design is promising for development as a versatile platform for enzyme immobilization.

Classification of Northeast China Cold Vortex Activity Paths in Early Summer Based on K-means Clustering and Their Climate Impact

The classification of the Northeast China Cold Vortex(NCCV)activity paths is an important way to analyze its characteristics in detail.Based on the daily precipitation data of the northeastern China(NEC)region,and the atmospheric circulation field and temperature field data of ERA-Interim for every six hours,the NCCV processes during the early summer(June)seasons from 1979 to 2018 were objectively identified.Then,the NCCV processes were classified using a machine learning method(k-means)according to the characteristic parameters of the activity path information.The rationality of the classification results was verified from two aspects,as follows:(1)the atmospheric circulation configuration of the NCCV on various paths;and(2)its influences on the climate conditions in the NEC.The obtained results showed that the activity paths of the NCCV could be divided into four types according to such characteristics as the generation origin,movement direction,and movement velocity of the NCCV.These included the generation-eastward movement type in the east of the Mongolia Plateau(eastward movement type or type A);generation-southeast longdistance movement type in the upstream of the Lena River(southeast long-distance movement type or type B);generationeastward less-movement type near Lake Baikal(eastward less-movement type or type C);and the generation-southward less-movement type in eastern Siberia(southward less-movement type or type D).There were obvious differences observed in the atmospheric circulation configuration and the climate impact of the NCCV on the four above-mentioned types of paths,which indicated that the classification results were reasonable.

Ultrasound-promoted Lipase-catalyzed Enantioselective Transesterification of (R,S)-Glycidol

Enantioselective transesterification of glycidol with vinyl butyrate as an acyl donor was investigated in the presence of Bacillus subtilis lipase（BSL2） as catalyst. Comparison studies demonstrate the advantage of ultrasound over the conventional shaking for the enzymatic reaction in non-aqueous media. The effects of reaction conditions（ultrasound power, temperature, water activity and pH） on the activity and enantioselectivity were also investigated. Under the optimum conditions, the synthetic activity of BSL2 was 2.95 μmol？min？1？mg？1 and the enantioselectivity（E value） was 52.2. Compared with conventional shaking, ultrasound made the synthetic activity and the enantioselectivity increase 9.5-fold and 1.4-fold, respectively. Furthermore, the repeated use of BSL2 for five cycles resulted in no obvious loss of enzyme activity, suggesting that the enzyme is stable under low power ultrasound conditions.