脂肪酶研发态势的全球专利分析

脂肪酶作为一种能够催化各种酯键断裂和形成的绿色环保生物催化剂,已被广泛应用于食品、饲料、洗涤剂、制药、精细化学以及生物修复等领域。基于智慧芽数据库（Pat Snap）对2020年10月前全球范围内各个国家及地区的脂肪酶相关专利进行统计,分析脂肪酶的技术分布、发展状况和未来趋势,并对比分析我国的脂肪酶研发现状,以期为脂肪酶技术及产业发展方向提供参考依据。     

Triton X-114 phase separation in the isolation and purification of mouse liver microsomal membrane proteins

Integral membrane proteins (IMPs) mediate several cellular functions including cell adhesion, ion and nutrient transport, and cell signalling. IMPs are typically hard to isolate and purify due to their hydrophobic nature and low cellular abundance, however, microsomes are small lipid vesicles rich in IMPs, which form spontaneously when cells are mechanically disrupted. In this study, we have employed mouse liver microsomes as a model for optimising a method for IMP isolation and characterisation. Microsomes were collected by differential centrifugation, purified with sodium carbonate, and subjected to GeLC–MS/MS analysis. A total of 1124 proteins were identified in the microsome fraction, with 47% (524/1124) predicted by TMHMM to contain at least one transmembrane domain (TMD). The ability of phase partitioning using the detergent Triton X-114 (TX-114) to further enrich for membrane proteins was evaluated. Microsomes were subjected to successive rounds of solubility-based phase separation, with proteins partitioning into the aqueous phase, detergent phase, or TX-114-insoluble pellet fraction. GeLC–MS/MS analysis of the three TX-114 fractions identified 1212 proteins, of which 146 were not detected in the un-fractionated microsome sample. Conspicuously, IMPs partitioned to the detergent phase, with 56% (435/770) of proteins identified in that fraction containing at least one TMD. GO Slim characterisation of the microsome proteome revealed enrichment of proteins from the endoplasmic reticulum, mitochondria, Golgi apparatus, endosome, and cytoplasm. Further, enzymes including monooxygenases were well represented with 35 cytochrome P450 identifications (CYPs 1A2, 2A5, 2A12, 2B10, 2C29, 2C37, 2C39, 2C44, 2C50, 2C54. 2C67, 2C68, 2C70, 2D10, 2D11, 2D22, 2D26, 2D9, 2E1, 2F2, 2J5, 2U1, 3A11, 3A13, 3A25, 4A10, 4A12A, 4A12B, 4F13, 4F14, 4F15, 4V3, 51,7B1, and 8B1). Evaluation of biological processes showed enrichment of proteins involved in fatty acid biosynthesis and elongation, as well as steroid synthesis. In addition, transport proteins including 24 members of the Rab family of GTPases were identified. Comparison of this dataset with the current mouse liver microsome proteome contributes an additional 648 protein identifications, of which 50% (326/648) contain at least one TMD.     

Solubilization and Structural Stability of Bacteriorhodopsin with a Mild Nonionic Detergent,n-Octyl-β-thioglucoside

Solubilization and structural stability of a membrane protein bacteriorhodopsin (bR) with n-octyl-β-thioglucoside (OTG) was investigated in comparison with a previous study on bR solubilized with n-octyl-β-glucoside (OG). Highly efficient and stable solubilization of bR with OTG was accomplished above the OTG concentration of about 15 mM. In comparison with OG-solubilized bR, the structural stability of OTG-solubilized bR was high in the dark and under light illumination. These results indicate that OTG is a detergent superior to OG for solubilizing bR molecules.     

Development of an Effective Sample Preparation Method for the Proteome Analysis of Body Fluids Using 2-D Gel Electrophoresis

A sample preparation is still the most critical step in two-dimensional electrophoresis (2-DE) and should be optimized for each type of sample. In this study, a protein extraction method from body fluids was developed using a combined centrifugal filter device and a sample treating buffer. When plasma, amniotic fluid, urine, and tear were tested with this method, the recovery of protein reached almost 90% and high-quality separation of 2-DE gel was obtained.     

Cholesterol depletion modulates detergent resistant fraction of human serotonin1A receptors

Abstract

Insolubility of membrane components in non-ionic detergents such as Triton X-100 at low temperature is a widely used biochemical criterion to identify, isolate and characterize membrane domains. In this work, we monitored the detergent insolubility of the serotonin_1A receptor in CHO cell membranes and its modulation by membrane cholesterol. The serotonin_1A receptor is an important member of the G-protein coupled receptor family. It is implicated in the generation and modulation of various cognitive, behavioral and developmental functions and serves as a drug target. Our results show that a significant fraction (～ 28%) of the serotonin_1A receptor resides in detergent-resistant membranes (DRMs). Interestingly, the fraction of the serotonin_1A receptor in DRMs exhibits a reduction upon membrane cholesterol depletion. In addition, we show that contents of DRM markers such as flotillin-1, caveolin-1 and GM₁ are altered in DRMs upon cholesterol depletion. These results assume significance since the function of the serotonin_1A receptor has previously been shown to be affected by membrane lipids, specifically cholesterol. Our results are relevant in the context of membrane organization of the serotonin_1A receptor in particular, and G-protein coupled receptors in general.     

Microbial inoculant effects on silage and in vitro ruminal fermentation, and microbial biomass estimation for alfalfa, bmr corn, and corn silages

Whole crop third cut alfalfa, brown mid-rib (bmr) corn, and corn were chopped and inoculated with one of four microbial inoculants used. Uninoculated silage was the control treatment. Each crop was ensiled in four mini-silos (1 L glass jars) per treatment. All silos were fermented for 60 days at room temperature (22 °C), and then they were opened and analyzed for fermentation products, fiber constituents and N fractions. A fraction of wet silage was ground with a blender for 30 s. In vitro gas production was measured in 160 ml sealed serum vials at 3, 6, 9, 24, and 48 h using the wet ground silage. At 9 and 48 h, rumen fluid was analyzed for volatile fatty acids (VFA) and microbial biomass yield (MBY). In all the three crops, the four inoculants produced only minor changes in pH and fermentation products during ensiling. Of the variables measured, soluble nonprotein N fractions were the characteristics most often affected by some inoculants. At 9 h incubation, in vitro gas production and VFA did not differ between control and inoculated silages, but MBY did. Among crops, alfalfa and corn silages had higher MBY than did bmr corn silage. Among inoculants, three of the four inoculated silages produced more MBY than did control. At 48 h, alfalfa silage produced higher MBY than did corn or bmr silage, and two of the inoculated silages had more MBY than did the control. There was no inoculant by crop interaction. Results suggest that some silage inoculants are capable of altering rumen fermentation, even in cases where effects on silage fermentation are small, and that this effect may be linked to better preservation of crop protein during ensiling.     

A pathway for protons in nitric oxide reductase from Paracoccus denitrificans

Nitric oxide reductase (NOR) from P. denitrificans is a membrane-bound protein complex that catalyses the reduction of NO to N₂O (2NO + 2e⁻ + 2H⁺ → N₂O + H₂O) as part of the denitrification process. Even though NO reduction is a highly exergonic reaction, and NOR belongs to the superfamily of O₂-reducing, proton-pumping heme-copper oxidases (HCuOs), previous measurements have indicated that the reaction catalyzed by NOR is non-electrogenic, i.e. not contributing to the proton electrochemical gradient. Since electrons are provided by donors in the periplasm, this non-electrogenicity implies that the substrate protons are also taken up from the periplasm. Here, using direct measurements in liposome-reconstituted NOR during reduction of both NO and the alternative substrate O₂, we demonstrate that protons are indeed consumed from the ‘outside’. First, multiple turnover reduction of O₂ resulted in an increase in pH on the outside of the NOR-vesicles. Second, comparison of electrical potential generation in NOR-liposomes during oxidation of the reduced enzyme by either NO or O₂ shows that the proton transfer signals are very similar for the two substrates proving the usefulness of O₂ as a model substrate for these studies. Last, optical measurements during single-turnover oxidation by O₂ show electron transfer coupled to proton uptake from outside the NOR-liposomes with a τ = 15 ms, similar to results obtained for net proton uptake in solubilised NOR [U. Flock, N.J. Watmough, P. Ädelroth, Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen, Biochemistry 44 (2005) 10711-10719]. NOR must thus contain a proton transfer pathway leading from the periplasmic surface into the active site. Using homology modeling with the structures of HCuOs as templates, we constructed a 3D model of the NorB catalytic subunit from P. denitrificans in order to search for such a pathway. A plausible pathway, consisting of conserved protonatable residues, is suggested.     

Lipid rafts and membrane traffic

Membrane rafts are regions of increased lipid acyl chain order that differ in their lipid and protein composition from the surrounding membrane. By providing an additional level of compartmentalization they have been proposed to serve many functions in cellular signal transduction and trafficking. We will review their potential involvement in different forms of membrane traffic, explicitly excluding signalling, and discuss select aspects of the raft hypothesis in its current form.     

Effect of zinc supplementation from two sources on growth,nutrient utilization and immune response in male crossbred cattle (Bos indicus×Bos taurus) bulls

To investigate effects of Zn supplementation on performance, mineral balance and immune response, 15 male crossbred cattle (Bos indicus×Bos taurus) bulls of about 14 ± 0.4 months of age and weighing 226.0 ± 9.1 kg were divided in to three groups of five. Bulls in the control group were fed wheat straw and a concentrate mixture (basal diet with 32.5 mg Zn/kg dry matter (DM)), and in ZnSO₄ and ZnProp groups 35 mg Zn/kg DM was added through Zn sulphate and Zn propionate, respectively. All bulls were fed their respective treatment diets for 180 days. Daily feed intake was recorded and bulls were weighed at every 15 days to determine change in body weight (BW). After 120 days of feeding, bulls were vaccinated with Brucella abortus strain 19, and cell mediated and humoral immune responses were assessed between 120 and 148 days of experimental feeding. After 150 days of feeding, a metabolism study of 6 days duration was completed to determine nutrient digestibility and mineral balances (i.e., Ca, P, Zn, Cu, Fe and Mn). Intake of total DM, digestibility of DM, crude protein, ether extract, neutral detergent fibre and acid detergent fibre, N balance, average daily gain, feed: gain did not differ between the groups. Intake, excretion and balance of Ca, P, Zn, Cu, Fe and Mn also did not differ between the groups. However, retention of Zn was higher (P<0.001) in the ZnProp group. Bulls supplemented with Zn propionate had higher cell mediated (P<0.01) and humoral (P<0.05) immune response, while there was no alteration in immune response due to Zn sulphate supplementation. Results indicate that a diet containing about 32.5 mg Zn/kg DM was adequate to support normal growth and digestibility, but a better immune response occurred when Zn propionate was added to the diet at 35 mg/kg DM versus Zn sulphate.     

A high-throughput method for membrane protein solubility screening: the ultracentrifugation dispersity sedimentation assay

One key to successful crystallization of membrane proteins is the identification of detergents that maintain the protein in a soluble, monodispersed state. Because of their hydrophobic nature, membrane proteins are particularly prone to forming insoluble aggregates over time. This nonspecific aggregation of the molecules reduces the likelihood of the regular association of the protein molecules essential for crystal lattice formation. Critical buffer components affecting the aggregation of membrane proteins include detergent choice, salt concentration, and presence of glycerol. The optimization of these parameters is often a time- and protein-consuming process. Here we describe a novel ultracentrifugation dispersity sedimentation (UDS) assay in which ultracentrifugation of very small (5 microL) volumes of purified, soluble membrane protein is combined with SDS-PAGE analysis to rapidly assess the degree of protein aggregation. The results from the UDS method correlate very well with established methods like size-exclusion chromatography (SEC), while consuming considerably less protein. In addition, the UDS method allows rapid screening of detergents for membrane protein crystallization in a fraction of the time required by SEC. Here we use the UDS method in the identification of suitable detergents and buffer compositions for the crystallization of three recombinant prokaryotic membrane proteins. The implications of our results for membrane protein crystallization prescreening are discussed.