兼具SOD和GPX活力的双功能酶的制备及性质研究

用苯甲基磺酰氟（ＰＭＳＦ）和Ｈ_２Ｓｅ相继处理铜锌超氧化物岐化酶（Ｃｕ,Ｚｎ－ＳＯＤ）,将酶分子中的丝氨酸（Ｓｅｒ）转化为硒代半胱氨酸（ＳｅＣｙｓ）,从而引入了谷胱甘肽过氧化物酶（ＧＰＸ）的催化基团,使其在ＳＯＤ酶活性大部分保留的情况下,具有ＧＰＸ活性,其ＧＰＸ活力是ＰＺ５１活力的３０倍。研究了双功能酶的最佳制备条件,包括ＰＭＳＦ的剂量、反应最适温度及Ｈ_２Ｓｅ处理时间等,并用电子能谱、ＤＴＮＢ等方法测定了双功能酶的硒含量;测定了双功能酶对不同底物的米氏常数及双功能酶的荧光光谱、紫外吸收光谱及稳定性。     

Conformation and Surface Properties of Deamidated Gluten

The surface properties of deamidated gluten were investigated with respect to their conformational changes. The helix content of gluten decreased curvilinearly with its decrease of deamidation. The surface tension decreased in proportion to the degree of deamidation. On the other hand, the surface hydrophobicity of gluten increased remarkably in proportion to the degree of deamidation. The emulsifying properties of gluten were improved greatly by deamidation, correlating linearly with the surface hydrophobicity. From these results, the relationships between the conformational changes and functional properties of deamidated gluten are discussed.     

Structure and Mechanism of Sanguinarine Reductase,an Enzyme of Alkaloid Detoxification

Sanguinarine reductase is a plant enzyme that prevents the cytotoxic effects of benzophenanthridine alkaloids, which are the main phytoalexins of Papaveraceae. The enzyme catalyzes the reduction of sanguinarine, the most toxic benzophenanthridine, which re-enters the cytoplasm after its primary accumulation in the cell wall region has reached a threshold concentration. We present the sequence of the gene and protein of sanguinarine reductase isolated from cell cultures of Eschscholzia californica. High sequence similarities indicate that the enzyme evolved from a plant-specific branch of the ubiquitous Rossmann fold NAD(P)H/NAD(P)⁺ binding reductases, with NADP-dependent epimerases or hydroxysteroid reductases as the most likely ancestors. Based on the x-ray structure of a close homolog, a three-dimensional model of the spatial conformation and catalytic site of sanguinarine reductase was established and used for in silico screening of known three-dimensional structures. Surprisingly, the enzyme shares high structural similarity with enzymes of human and bacterial origin, which have similar functions as the plant homologs but bear little amino acid sequence similarity. Using site-directed mutagenesis, a series of recombinant enzymes was generated and assayed to reveal the impact of individual amino acids and peptides in the catalytic process. It appears that relatively few innovations were required to generate this selective catalyst for alkaloid detoxication, notably an insertion of 13 amino acids and the generation of a novel catalytic triad of Cys-Asp-His were sufficient.     

Ineffective Degradation of Immunogenic Gluten Epitopes by Currently Available Digestive Enzyme Supplements

Background

Due to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP).

Methods

Five commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1) enzyme assays and 2) mass spectrometric identification. Gluten epitope degradation was monitored by 1) R5 ELISA, 2) mass spectrometric analysis of the degradation products and 3) T cell proliferation assays.

Findings

The digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data.

Conclusion

Currently available digestive enzyme supplements are ineffective in degrading immunogenic gluten epitopes.     

A Food-Grade Cloning System for Industrial Strains of Lactococcus lactis

We have previously reported the construction of a food-grade cloning vector for Lactococcus using the ochre suppressor, supB, as the selective marker. This vector, pFG1, causes only a slight growth inhibition in the laboratory strain MG1363 but is unstable in the industrial strains tested. As supB suppresses both amber and ochre stop codons, which are present in 82% of all known lactococcal genes, this undesirable finding may result from the accumulation of elongated mistranslated polypeptides. Here, we report the development of a new food-grade cloning vector, pFG200, which is suitable for overexpressing a variety of genes in industrial strains of Lactococcus lactis. The vector uses an amber suppressor, supD, as selectable marker and consists entirely of Lactococcus DNA, with the exception of a small polylinker region. Using suppressible pyrimidine auxotrophs, selection and maintenance are efficient in any pyrimidine-free medium including milk. Importantly, the presence of this vector in a variety of industrial strains has no significant effect on the growth rate or the rate of acidification in milk, making this an ideal system for food-grade modification of industrially relevant L. lactis strains. The usefulness of this system is demonstrated by overexpressing the pepN gene in a number of industrial backgrounds.     

Preparation of ADP-Glucose with an Enzyme from Arthrobacter simplex

For the purpose of enzymatic preparation of ADP-glucose (ADPG), bacterial screening was performed to find a strain having a high activity of ADPG pyrophosphorylase which catalyzes the synthesis of ADPG from ATP and glucose-1-phosphate. A cell-free extract of Arthrobacter simplex IFO 12069 showed a strong enzyme activity for the synthesis of ADPG, which was isolated from the reaction solution by ion-exchange column chromatography and identified by paper and thin-layer chromatography. The enzyme activity of the bacterium reached a maximum in the late logarithmic phase under aerobic growth conditions. Some factors affecting the ADPG synthesis, e.g. reaction pH, substrate concentrations, divalent cations, inhibitors and activators, were studied with an ammonium sulfate fraction, 30~50% saturation as the enzyme preparation.     

Modest Influence of FRET Chromophores on the Properties of Unfolded Proteins

Single-molecule Förster resonance energy transfer (FRET) experiments are often used to study the properties of unfolded and intrinsically disordered proteins. Because of their large extinction coefficients and quantum yields, synthetic heteroaromatic chromophores covalently linked to the protein are often used as donor and acceptor fluorophores. A key issue in the interpretation of such experiments is the extent to which the properties of the unfolded chain may be affected by the presence of these chromophores. In this article, we investigate this question using all-atom explicit solvent replica exchange molecular dynamics simulations of three different unfolded or intrinsically disordered proteins. We find that the secondary structure and long-range contacts are largely the same in the presence or absence of the fluorophores, and that the dimensions of the chain with and without chromophores are similar. This suggests that, at least in the cases studied, extrinsic fluorophores have little effect on the structural properties of unfolded or disordered proteins. We also find that the critical FRET orientational factor κ², has an average value and equilibrium distribution very close to that expected for isotropic orientations, which supports one of the assumptions frequently made when interpreting FRET efficiency in terms of distances.     

Modest Influence of FRET Chromophores on the Properties of Unfolded Proteins

Single-molecule Förster resonance energy transfer (FRET) experiments are often used to study the properties of unfolded and intrinsically disordered proteins. Because of their large extinction coefficients and quantum yields, synthetic heteroaromatic chromophores covalently linked to the protein are often used as donor and acceptor fluorophores. A key issue in the interpretation of such experiments is the extent to which the properties of the unfolded chain may be affected by the presence of these chromophores. In this article, we investigate this question using all-atom explicit solvent replica exchange molecular dynamics simulations of three different unfolded or intrinsically disordered proteins. We find that the secondary structure and long-range contacts are largely the same in the presence or absence of the fluorophores, and that the dimensions of the chain with and without chromophores are similar. This suggests that, at least in the cases studied, extrinsic fluorophores have little effect on the structural properties of unfolded or disordered proteins. We also find that the critical FRET orientational factor κ², has an average value and equilibrium distribution very close to that expected for isotropic orientations, which supports one of the assumptions frequently made when interpreting FRET efficiency in terms of distances.     

Isolation and Properties of Carbohydrate Rich Fraction of Wheat Gluten

Two carbohydrate rich fractions A and B were isolated from wheat gluten. Fraction B contained more lipid than fraction A. Lipid portion of fraction B consisted mainly of glycolipid and was fractionated into five fractions by thin-layer chromatography. The two main fractions were extracted and determined to be galactolipid and glucolipid, respectively, by the analyses of fatty acid and sugar components by gas chromatography. Defatted fraction A was assumed to consist of glycoprotein. After complete pronase digestion of defatted fraction A, the remaining glycopeptide moiety was isolated by column chromatography on DEAE-cellulose followed by gel filtration through Sephadex G–25. The amino acid and sugar components of the glycopeptide were investigated.     

A Ferritin from Dendrorhynchus zhejiangensis with Heavy Metals Detoxification Activity

Ferritin, an iron homeostasis protein, has important functions in transition and storage of toxic metal ions. In this study, the full-length cDNA of ferritin was isolated from Dendrorhynchus zhejiangensis by cDNA library and RACE approaches. The higher similarity and conserved motifs for ferritin were also identified in worm counterparts, indicating that it belonged to a new member of ferritin family. The temporal expression of worm ferritin in haemocytes was analyzed by RT-PCR, and revealed the ferritin could be induced by Cd²⁺, Pb²⁺ and Fe²⁺. The heavy metal binding activity of recombinant ferritin was further elucidated by atomic force microscopy (AFM). It was observed that the ferritin protein could form a chain of beads with different size against three metals exposure, and the largest one with 35∼40 nm in height was identified in the Cd²⁺ challenge group. Our results indicated that worm ferritin was a promising candidate for heavy metals detoxification.     

Properties of an Immobilized Pesticide-Hydrolyzing Enzyme

A bacterial enzyme(s) capable of hydrolyzing nine organophosphate insecticides was covalently bound to glass. The efficiency of this binding reaction ranged from 4 to 17%. Under continuous column operation, the immobilized enzyme(s) had an extrapolated half-life of 280 days. The specific activity of this glass-covalently bound hydrolase activity for parathion varied from 0.035 to 0.15 μmol/min per g of glass. The bound activity increased with decreasing glass particle size; however, the flow resistance also increased. Immobilized enzyme(s) kinetics were approximately 50% slower than those of the free enzyme(s), but there was no significant difference in the effect pH and temperature had on the activity of immobilized and free enzyme(s).     

A Nonproteolytic "Trypsin-like" Enzyme: Purification and Properties of Arachain

By the use of the proteolytic substrates benzoyl-dl-arginine-p-nitroanilide and benzoyl-l-arginine ethyl ester the enzyme arachain has been purified 325-fold from acetone powders of ungerminated peanuts. The pH optimum for the hydrolysis of benzoyl-dl-arginine-p-nitroanilide was 8.1 in tris buffer, and for benzoyl-l-arginine ethyl ester was 7.5 using N - 2 - hydroxyethylpiperazine - N′ - 2 - ethanesulfonic acid buffer. The purest fraction showed one main band with one to three minor bands on disc gel electrophoresis. The major protein component had an S_20,w of 6.20. The energy of activation for the hydrolysis of benzoyl-dl-arginine-p-nitroanilide was calculated to be 16 kilocalories. The Michaelis constant for benzoyl-dl-arginine-p-nitroanilide was 10 micromolar and for benzoyl-l-arginine ethyl ester was 110 micromolar. The enzyme showed essentially no activity with casein, dimethyl casein, or bovine serum albumin as substrates. A large number of peptides were hydrolyzed by the enzyme, only l-leucyl-l-tyrosine being resistant of the peptides tested. The results suggest that arachain is not a “trypsin-like” protease but is a peptide hydrolase.     

Promoting More Modest Weight Losses: A Pilot Study

Objective: This pilot study assessed the short‐ and long‐term effects of a modified cognitive behavioral treatment designed to facilitate obese patients’ acceptance of a 5% to 10% reduction in initial weight. Research Methods and Procedures: Participants were 17 women with a mean age of 46.5 ± 9.7 years and BMI of 34.7 ± 2.9 kg/m². They participated in a 40‐week program that included four phases. The first discussed the benefits of modest weight losses and the potential adverse effects of unrealistic expectations. Phase II provided instruction in traditional cognitive behavioral methods of weight control Phase III focused on methods to improve body image and self‐esteem. Phase IV addressed skills for weight maintenance. Changes in weight, self‐esteem, body image, and quality of life were assessed at the end of treatment and 1 year later (week 92). Results: At week 40, participants lost an average of 5.7 ± 5.3% of initial weight, which was associated with significant improvements in body image, self‐esteem, and quality of life. Improvements in psychosocial status were maintained at week 92, although mean weight loss at this time had declined to 2.9 ± 5.6% of initial weight. Increased satisfaction with body weight at week 40 was associated with significantly better maintenance of weight loss at follow‐up (r = ?0.70; p = 0.02). Discussion: Having participants seek only modest initial weight losses does not appear to facilitate weight maintenance. However, increasing patients’ satisfaction with their body weight at the end of treatment may help improve weight maintenance. More research is needed on the relation between satisfaction with initial weight loss and long‐term success.     

双酶法制备螺旋藻多肽的工艺研究

选用碱性蛋白酶和木瓜蛋白酶结合的双酶法对螺旋藻蛋白进行水解。其中,对木瓜蛋白酶水解螺旋藻蛋白的工艺进行优化。以水解度为指标,研究了酶解时间、酶与底物比、pH和酶解温度4种因素对酶解反应的影响。在此基础上设计了3因素(加酶量、酶解温度和pH)3水平的响应面试验。结果表明碱性蛋白酶水解螺旋藻蛋白的最佳酶解条件为:加酶量4300 U/g,pH 7.0,酶解温度55℃,酶解时间160 min;木瓜蛋白酶的最佳酶解条件为:酶底比为4.5%,酶解温度60℃,pH 6.5,酶解时间210 min。利用碱性蛋白酶和木瓜蛋白酶结合的双酶法制得的多肽水解度可达32.90%,与单酶法相比,水解度明显提高。     

酶法拆分制备D-异亮氨酸的工艺研究

以DL-异亮氨酸为原料经过乙酰化、氨基酰化酶拆分和水解制备D-异亮氨酸.使乙酸酐与DL-异亮氨酸在0～10℃下反应生成乙酰-DL-异亮氨酸,在氨基酰化酶的作用下,温度为37℃搅拌反应3d处理得到L-异亮氨酸和乙酰-D-异亮氨酸,乙酰-D-异亮氨酸经过盐酸水解得到D-异亮氨酸,收率为97.4％,光学纯度达到98％以上.另...     

采用诱导环化酶制备Illumina Mate-paired DNA文库

新一代测序技术(NGS)的文库制备方法在基因组的拼装中起着重要作用。但是NGS技术制备的普通DNA文库片段只有500 bp左右,难以满足复杂基因组的从头(de novo)拼装要求。三代测序技术的读长可以达到20 kb,但是其高错误率及测序成本过高使得其又不易推广。因此二代测序的Mate-paired文库制备技术一直在基因组的de novo拼装中扮演着非常重要的角色。目前主流的NGS平台Illumina制备的Mate-paired文库的片段范围只有2~5 kb,为了得到更长的可用于Illumina平台测序的Mate-paired文库,本研究首次整合并优化了Illumina和Roche/454两种测序平台的Mate-paired文库制备技术,采用诱导环化酶来提高基因组长片段DNA的环化效率,成功建立了20 kb Mate-paired文库制备技术,并已将该技术应用于人类基因组20 kb Mate-paired文库制备。该技术为Illumina平台制备长片段Mate-paired库提供了方法指导。     

甘油三酯酶传感器的研制及应用

将脂肪酶固定在pH玻璃电极上,制成甘油三酯酶传感器.对传感器的制作方法和响应性能进行了研究.在37℃,选择Tris-HCl缓冲溶液(pH 8.5)为测量介质, 所制传感器对甘油三酯的响应线性范围为3.09×10^－6～1.91×10^－3 mol/L,响应斜率为32.7 mV/pC, 响应时间为5～10 min, 使用寿命可达25 d.用研制的传感器测定了人和兔血清中的甘油三酯含量,结果满意.     

Lysinoalanine Formation in Gluten: A Conformational Effect

The production of pectolytic enzyme in the genus Kluyveromyces was investigated. The production of the enzyme was dependent on the strain, and some strains belonging to K. fragilis, K. Marxianus, and K. Wickerhamii produced this enzyme among 11 species (29 strains) of the genus Kluyveromyces. K. Fragilis IFO 0288 produced at least four endo-polygalacturonases which have different molecular weights. The dominant endo-polygalacturonase in the culture filtrate of the strain was purified and isolated as crystals. The purified enzyme was homogeneous based on analysis by polyacrylamide gel electrophoresis and ultracentrifugation. The enzyme was a glycoprotein having an isoelectric point around pH 5.6. The sedimentation coefficient (s_2o,w) was 3.77S, and the molecular weight was around 33,000. The enzyme contained aspartic acid (asparagine), serine,threonine, and glycine at relatively high levels. The enzyme showed the highest activity around pH 5.0 and was stable at pH 5.0 up to 30°C. With the enzyme, and activity which releases highly polymerized pectin from various protopectins (protopectinase activity) was found.     

A Food-Grade Approach for Functional Analysis and Modification of Native Plasmids in Lactococcus lactis

While plasmids from lactic acid bacteria possess many traits that are of industrial value, their exploitation is often frustrated by an inability to conduct food-grade engineering of native plasmids or to readily screen for their transfer. Here we describe a system that uses a RepA⁺ temperature-sensitive helper plasmid and a RepA⁻ cloning vector to overcome these problems while maintaining the food-grade status of the native plasmid. This strategy was used to precisely delete ltnA1 alone, or in conjunction with ltnA2 (encoding the structural proteins of the lantibiotic lacticin 3147), from the native 60.2-kb plasmid pMRC01 and to select for the transfer of pMRC01 between Lactococcus lactis strains.     

A Model Transgenic Cereal Plant with Detoxification Activity for the Estrogenic Mycotoxin Zearalenone

Zearalenone (ZEN) is an estrogenic mycotoxin produced by the necrotrophic cereal pathogen Fusarium graminearum. This mycotoxin is detoxified by ZHD101, a lactonohydrolase from Clonostachys rosea, or EGFP:ZHD101, its fusion to the C-terminus of an enhanced green fluorescence protein. We previously showed that egfp:zhd101 is efficiently expressed in T₀ leaves of rice. In this study, we assessed the feasibility of in planta detoxification of the mycotoxin using progeny. When protein extract from T₁ leaves was incubated with ZEN, the amount of the toxin decreased significantly as measured by HPLC. ZEN degradation activity was also detected in vivo in transgenic T₂ seeds. These results suggest that zhd101 can be exploited as an efficient and cost-effective system for protection of important cereals that are more susceptible to the pathogen (e.g., wheat and maize) from contamination with the estrogenic mycotoxin.