Extraction of Natural Nanostructured Hydroxyapatite from Pacific Cod(Gadus macrocephalus)Bone with a Thermostable Collagenolytic Protease and Its ex vivo Intestinal Bioavailability

Natural nano-hydroxyapatite(HA)was extracted from Pacific cod(Gadus macrocephalus)bone with a thermostable col-lagenolytic protease in the present study.Conditions for the enzymatic reaction were optimized to be 60℃and pH 7.0,and a desir-able extraction efficiency was achieved by using the crude collagenolytic protease.Dynamic light scattering,transmission electron microscopy and energy-dispersive X-ray analysis revealed that nano-HA are anionic spherical(about 110nm)particles mainly com-prised of calcium and phosphorus at an approximate ratio of 5:3.As evaluated with the mouse ex vivo intestinal segments,the extracted nano-HA displayed comparable level of intestinal bioavailability to the positive control CaCl_(2).By treating with inhibitors(NaN3,ami-loride)and low temperature(4℃),clathrin-mediated endocytosis was assumed to involve the intestinal absorption of nano-HA.Over-all,the application of thermostable collagenolytic protease is proved to be a promising alternative method for nano-HA extraction from natural resource with improved ecological and biological value.

Thermostable ethanol tolerant xylanase from a cold-adapted marine species Acinetobacter johnsonii

A xylanase-producing bacterium, isolated from deep sea sediments, was identified as the cold-adapted marine species Acinetobacter Johnsonii. A cold-adapted marine species Acinetobacter Johnsonii could grow at 4 ℃. The optimum temperature and pH of xylanase from a cold-adapted marine species Acinetobacter Johnsonii were 55 ℃ and pH 6.0. Xylanase from a cold-adapted marine species Acinetobacter Johnsonii remained at 80% activity after incubation for 1 h at 65 ℃. The xylanase activity was 1.2-fold higher in 4% ethanol solution than in ethanol free solution. Gibbs free energy of denaturation, ΔG, was higher in 4% ethanol solution than in ethanol free solution. Thermostable ethanol tolerant xylanase was valuable for bioethanol production by simultaneous saccharification and fermentation process with xylan as a carbon source.

Purification and Characterization of a Novel Thermostable Chitinase from Thermomyces lanuginosus SY2 and Cloning of Its Encoding Gene

A novel thermostable extracellular chitinase was purified from the culture filtrate of Thermomyces lanuginosus SY2 by using diethylaminoethyl Sepharose chromatography and Phenyl-Sepharose chromatography. The molecular size of the purified chitinase was estimated to be 48 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chitinase exhibited optimum catalytic activity at pH 4.5 and 55℃. The enzyme was stable at 50℃, and its half-life time at 65℃ was 25 rain. The thermostable chitinase was obtained with 60% of the full activity, when it was incubated in the buffer （pH 2.5）. The enzyme showed the unique properties for thermostability and pH stability since it was one of the most thermostable chitinases so far isolated in fungi. Ca^2＋, Ba^2＋, Na^＋, and K^＋ enhanced the enzyme activity, whereas Fe^2＋, Ag^＋, Hg^2＋, and ethylene diamine tetraacetic acid caused obvious inhibition. The N-terminal amino acids were AQGYLSVQYFVNWAI. Degenerate primers based on the N-terminal sequences of purified chitinase and a cDNA fragment encoding the chitinase gene were obtained through reverse transcriptase-polymerase chain reaction amplication. The RACE was used to generate full-length cDNA clones. The cDNA of chit contained an open reading frame of 1 326 bp encoding 442 amino acids. The gene chit has been registered in GenBank with accession number DQ092332. The alignment results of putative amino acid sequence showed the lower similarity to other chitinases in family-18 except for the catalytic domain containing two conserved motifs related with catalytic activity of chitinase.

Purification and Characterization of a New Thermostable κ-CarrageenasefromtheMarineBacterium Pseudoalteromonas sp. QY203

A new extracellular κ-carrageenase, namely CgkP, 34.0 kDa in molecular weight, was purified from Pseudoalteromonas sp. QY203. CgkP showed relatively high activity at acidities ranging from pH6.0 to pH9.0 and temperatures ranging from 30℃ to 50℃ with the highest activity at 45℃ and pH7.2. Sodium chloride increased its activity markedly, and KC1 increased its activity slightly. The divalent and trivalent metal ions including Cu^2＋, Ni^2＋, Zn^2＋, Mn^2＋, Al^3＋ and Fe^3＋ significantly inhibited its activity, while Mg^2＋ did not. CgkP remained 70% of original activity after being incubated at 40℃ for 48h, and remained 80% of the activity after being incubated at 45℃ for 1 h. It exhibited endo-κ-carrageenase activity, mainly depolymerizing the κ-carrageenan into disaccharide and tetrasaccharide. CgkP was more thermostable than most of previously reported κ-carrageenases with a potential of being used in industry.

Thermostable Broad Band Polarizing PVA-Film: Theoretical and Experimental Investigations

In the present work, for the first time on the basis ofpoly （vinyl alcohol） （PVA）, 2- （4-dimethylaminostyryl）-l-ethylquinolinium iodide （quinaldine red （QR）） and trisodium （4E）-5-oxo- 1-（4-sulfonatophenyl）-4-[（4-sulfonatophenyl）hydrazono]-3 pyrazolecarboxylate （tartrazine （T））, thermostable polarizing film in a wide range of spectra （λmax=394-511 nm） with polarization efficiency （PE） = 98% in absorption maximum and stretching degree （Rs） = 3.5 was developed. The basic spectral-polarization parameters （polarization efficiency and transmittance） of oriented colored PVA-films were measured and discussed. During the work it was found that oriented PVA-films are the phenomenon of anisotropy of thermal conductivity （λ｜/λ⊥）. It is a very important parameter for the development of thermostable PVA-polarizing films. For the first time quantum-chemical calculations using density functional theory （DFT） approach for structural analysis and electronic spectrum of the QR were carried out via the B3LYP/dgdzvp and TDB3LYP/dgdzvp methods. Interpretation of absorption strips in visible region of spectrum was also reported. The excitation energies, electronic transitions and oscillator strengths for the studied structures have also been calculated （B3LYP/dgdzvp）. The NBO analysis and Mulliken atomic charges of the QR were carried out.

Discovery and Characterization of a Thermostable Esterase from an Oil Reservoir Metagenome

With the aim of identifying novel thermostable esterases, comprehensive sequence databases and cloned fosmid libraries of metagenomes derived from an offshore oil reservoir on the Norwegian Continental Shelf were screened for enzyme candidates using both sequence-and function-based screening. From several candidates identified in both approaches, one enzyme discovered by the functional approach was verified as a novel esterase and subjected to a deeper characterization. The enzyme was successfully over-produced in Escherichia coli and was shown to be thermostable up to 90°C, with the highest esterase activity on short-chain ester substrates and with tolerance to solvents and metal ions. The fact that the thermostable enzyme was solely found by functional screening of the oil reservoir metagenomes illustrates the importance of this approach as a complement to purely sequence-based screening, in which the enzyme candidate was not detected. In addition, this example indicates the large potential of deep-sub-surface oil reservoir metagenomes as a source of novel, thermostable enzymes of potential relevance for industrial applications.

Characterization of a thermostable manganese-containing superoxide dismutase from inshore hot spring thermophile Thermus sp.JM1

A thermostable superoxide dismutase （SOD） from the inshore thermophile Thermus sp. JM1 was purified to homogeneity by steps of fractional ammonium sulfate precipitation, DEAE-Sepharose chromatography and Phenyl-Sepharose chromatography. The specific activity of the purified native enzyme was 1 656 U/mg. A sod gene from this strain was cloned and overexpressed in Escherichia coli （E. coli）. The prepared apo-enzyme of the purified recombinant SOD （rSOD） was reconstituted with either Fe or Mn by means of incubation with appropriate metal salts. As a result, only Mn 2＋ - reconstituted rSOD （Mn-rSOD） exhibited the specific activity of 1 598 U/mg. SOD from Thermus sp. JM1 was Mn-SOD, judging by the specific activities analysis of Fe or Mn reconstituted rSODs and the insensitivity of the native SOD to both cyanide and H 2 O 2 . Both the native SOD and Mn- rSOD were determined to be homotetramers with monomeric molecular mass of 26 kDa and 27.5 kDa, respectively. They had high thermostability at 50 ° C and 60 ° C, and showed striking stability across a wide pH span from 4.0 to 11.0.

Overexpression and characterization of a thermostable β-agarase producing neoagarotetraose from a marine isolate Microbulbifer sp.AG1

An agarase gene containing 1 302 bp was cloned from Microbulbifer sp. AG1. It encoded a mature protein of 413 amino acids plus a 20-residue signal peptide. The recombinant enzyme without the signal peptide was expressed and purified from Escherichia coli BL21(DE3). When agarose was used as a substrate, the optimal temperature and pH for the enzyme were 60℃ and 7.5, respectively. The recombinant agarase showed excellent thermostability with 67% and 19% of residual activities after incubation at 50℃ and 60℃ for 1 h, respectively.Except SDS, the recombinant agarase had a relatively good resistance against the detected inhibitors, detergents and urea denaturant. Thin layer chromatography analysis and enzyme assay using p-nitrophenyl-α/β-Dgalactopyranoside revealed that the recombinant agarase was a β-agarase that degraded agarose into neoagarotetraose as the main end product. The enzymatic hydrolysis products with different degree of polymerization exhibited the antioxidant activities.

Optimized production and properties of thermostable alkaline protease from Bacillus subtilis SHS-04 grown on groundnut (Arachis hypogaea) meal

Production of alkaline protease from Bacillus subtilis SHS-04 was investigated under different fermentation conditions involving low-cost substrates with the aim of optimizing yield of enzyme. Maximum enzyme production (1616.21 U/mL) was achieved using groundnut meal (0.75%) as nitrogen source and 0.5% glucose as carbon source at 48 h cultivation period, pH 9, 45 ° C and 200 rpm. The yield was 348% increase over comparable control samples. The alkaline protease had optimum temperature of 60 ° C and remarkably exhibited 80% relative activity at 70 ° C. It was highly thermostable showing 98.7% residual activity at 60 ° C after 60 minutes of incubation at pH 9.0 and was stable in the presence of organic solvents studied. These properties indicate the viability of the protease for biotechnological and industrial applications. The optimized yield of enzyme achieved in this study establishes groundnut meal as potential low-cost substrate for alkaline protease production by B. subtilis SHS-04.

Optimization of Growth Conditions to Identify the Superior Bacillus Strain Which Produce High Yield of Thermostable Alpha Amylase

Thermostable α-amylases hold a very important place in commercial industrial applications in Sri Lanka. Therefore, the main aim of this study was to identify superior Bacillus strain and optimize growth conditions that could yield high α-amylase production. Three Bacillus strains, B. amyloliquefaciens ATCC 23350, B. licheniformis ATCC 14580 and B. megaterium ATCC 14581 were used for the study. Shake flask culture experiments were conducted to identify the effect of various fermentation conditions such as growth temperature, incubation period, carbon source, nitrogen source, initial pH and carbon concentration on extracellular α-amylase production. DNSA assay was carried out to determine the enzyme activity. The highest temperature for enzyme activity was reported by B. licheniformis at 85°C, followed by B. amyloliquefaciens at 75°C and B. megaterium at 45°C. Both B. amyloliquefaciens and B. licheniformis were able to give their optimum enzyme production at 37°C, while B. megaterium at 30°C in 150 rpm with initial pH of 7. B. licheniformis and B. amyloliquefaciens gave their optimum yield of the enzyme after 48 h of incubation while B. megaterium gave after 24 h of incubation. Among the carbon sources tested cassava starch was able to give the highest enzyme production. For B. amyloliquefaciens, the highest yield of the enzyme was obtained with 2% of starch, tryptone as a nitrogen source and initial pH of 7. Maximum enzyme production for B. licheniformis was obtained with 1.5% of starch, KNO3 as a nitrogen source and initial pH of 6. For B. megaterium 1% of starch, tryptone and pH 7.5 induced the optimum α-amylase production. According to the results obtained, B. amyloliquefaciens is the highest thermostable alpha amylase producer. However, according to the industrial requirement, B. licheniformis can also be used as an enzyme producer due to its stability in higher temperatures.

A thermostable serralysin inhibitor from marine bacterium Flavobacterium sp. YS-80-122

Serralysin inhibitors have been proposed as potent drugs against many diseases and may help to prevent further development of antibiotic-resistant pathogenic bacteria. In this study, a novel serralysin inhibitor gene, lupI, was cloned from the marine bacterium Flavobacterium sp. YS-80-122 and expressed in Escherichia coll. The deduced serralysin inhibitor, LupI, shows 〈40% amino acid identity to other reported serralysin inhibitors. Multiple sequence alignment and phylogenetic analysis of LupI with other serralysin inhibitors indicated that LupI was a novel type of serralysin inhibitor. The inhibitory constant for LupI towards its target metalloprotease was 0.64mol/L. LupI was thermostable at high temperature, in which 35.6%-90.7% of its inhibitory activity was recovered after treatment at 100℃ for 1-60 min followed by incubation at 0℃. This novel inhibitor may represent a candidate drug for the treatment of serralysin-related infections.

Thermostable alkaline protease production from Bacillus pumilus D-6 by using agro-residues as substrates

Proteases due to their wide range of applications in biotechnological processes have been the??focus of intense research for many decades. However, from industrial?application view point most of the available proteases lack desired properties;?therefore, search for better and efficient thermostable alkaline proteases are?always on.?Bacillus pumilus?D-6, isolated from dairy plant soil sample, in the?current study produced protease which showed activity and stability at high?alkaline?pH (8 - 12) and high?temperatures (70。C- 100。C). Enzyme activity remained unfazed even in presence?of inhibitors like Pb2+and Hg2+which are considered?universal inhibitors of enzyme activity. Besides, the organism successfully?utilized crude agriculture based substrates as carbon and nitrogen source and?produced substantial enzyme titre.

Field Trial of a Thermostable Peste des petits ruminants (PPR) Vaccine in a Semi-Arid Zone of Nigeria

The field trial of a candidate thermostable Peste des petits ruminants (PPR) vaccine was carried out in flocks of sheep and goats under the extensive system of management. The immune response of vaccinated animals was determined using the neutralisation test to detect PPR virus specific antibody. Vaccinated animals seroconverted and a four-fold or more rise in antibody titre were observed between pre-vaccination and post-vaccination antibodies. The vaccine elicited significant antibody response in goats through the different routes of administration (intramuscular, intranasal, intraocular, subcutaneous and orally), but was poorly transmitted between the vaccinees and in-contact animals. The sheep responded poorly to the vaccine administered through most of the routes, except for those vaccinated through intramuscular and subcutaneous routes that seroconverted significantly (≥4 fold rise). The vaccine retained a potent titre of 3.1 log10 TCID50 for more than 8 hours after reconstitution in PBS at room temperature. Based on the response of goats to oral vaccination, it is suggested that the vaccine could be administered on the field through the oral routes and has the potential to be adapted to a feed-based administration for wider application to the scattered livestock populations under the extensive system of management.

祖先序列重建增强D-阿洛酮糖3-差向异构酶的热稳定性

为解决现有D-阿洛酮糖3-差向异构酶(DAEase)热稳定性差的产业问题,本文采用系统发育指导的大数据挖掘、合理修饰和祖先序列重建策略(ASR),重建了具有不同催化结构域DAEase的祖先序列,构建了表达载体,通过重组表达与分子对接筛选出了DAEase A13并进行酶学性质表征,此外,还基于结构分析与分子动力学模拟揭示了DAEase A13热稳定性增强的分子机制。结果表明,基于ASR策略所构建的A13 70℃时半衰期可达8.4 h,其热稳定性较野生(WT)酶显著增强,最大转化率为31%,催化活性也略高于WT酶。立体结构模拟与分子动力学模拟揭示了ASR A13中大量氢键和疏水作用的增加维持了高温下酶分子结构的稳定性,是其热稳定性增强的主要因素。研究结果证实了ASR策略可以改造DAEase使其稳定性、活性和混杂性增强,可以为D-阿洛酮糖工业生产提供良好的生物催化剂。

蛋白质分子表面氨基酸突变提高植酸酶Yi APPA的活性和热稳定性

为了提高植酸酶的活性和热稳定性,增加其在食品加工领域的应用潜力,对植酸酶Yi APPA进行同源模建,结合蛋白质分子表面氨基酸突变策略,选择位于分子表面的赖氨酸和甘氨酸进行定点突变,构建单位点突变体。通过活性和热稳定性筛选,获得活性和热稳定性显著提高的突变体K216R以及热稳定性提高的突变体K189R。通过有益突变位点叠加策略,进一步构建并表征组合突变体K189R/K216R的酶活力及热稳定性。结果表明:与Yi APPA相比,K189R/K216R于80℃半衰期由14.81 min延长至23.35 min,半失活温度由55.12℃提升至62.44℃,热解折叠温度由48.36℃提升至53.18℃。并且K189R/K216R于37℃、pH 4.5的酶活力由3959.98 U/mg提高至4469.13 U/mg。分子结构建模和分子动力学模拟的结果显示:K189R/K216R中引入了新的氢键,能够提高酶部分结构单元的稳定性,使其热稳定性得到提高;同时K189R/K216R的催化口袋体积增大是其活性提高的主要原因。本研究通过蛋白质分子表面氨基酸突变策略可有效提高植酸酶Yi APPA的活性和热稳定性,为植酸酶及其他类型酶的分子改造提供参考依据。

大米淀粉晶体/非晶体结构耐热性研究

采用中红外(MIR)光谱及二维中红外(2D-MIR)光谱分别开展大米结构及其淀粉晶体/非晶体结构热变性研究。实验发现:大米的红外吸收模式主要包括:νOH-大米、νas CH2-大米、νs CH2-大米、νamide-Ⅰ-大米、νamide-Ⅱ-大米和νC-O-大米。随着测定温度升高(303~393 K),不同产地的大米淀粉晶体/非晶体结构对于热的敏感程度及变化快慢顺序均存在一定的差异性。进一步探究相关机理,认为不同大米淀粉晶体/非晶体结构耐热性存在一定差异性,因而进一步影响其口感。并拓展了MIR光谱及2D-MIR光谱技术在大米结构及其淀粉晶体/非晶体结构热变性的研究范围。

高压电场选育高产酸性木聚糖酶菌株的研究

为获得适用于动物饲料的木聚糖酶高产菌株,并验证高压电场诱变方法的有效性,于农田土壤中筛选出高产菌株,在高压电场中进行诱变,筛选最佳的突变菌株。出发菌株初步鉴定为沙福芽孢杆菌,命名为Bacillus safensis B16。该菌株所产木聚糖酶在pH 5、60℃下酶活性为217.34 U/mL,且60℃时的半衰期为229 min,热稳定性良好。在6~24 kV、极间距为3 cm的高压电晕电场中处理0~10 min,最终在10 kV下处理6 min选育出一株木聚糖酶高产菌株B16106-2,酶活性达到256.73 U/mL,较出发菌株提升了18.12%,且传代7次后菌株的产酶能力基本不变,具有良好的遗传稳定性。突变菌株所产木聚糖酶的最适酶反应条件不变。研究证明,高压电晕电场是一种有效的诱变方式,且筛选出的菌株B16106-2性能良好。

基于宏基因组技术的生鲜乳中耐热酶污染来源分析

生鲜乳中的耐热酶会严重影响产品货架期的质量稳定性,而耐热酶的主要来源和污染途径一直存在争论。基于宏基因组技术分析中国某牧场的环境样本(牛舍垫土、TMR饲料)和生鲜乳样本(头三把奶、头三把奶之后生鲜乳、储奶罐中生鲜乳)的微生物群落组成及其产胞外蛋白酶(EC 3.4.24.40)和脂肪酶(EC 3.1.1.3)的基因丰度,弄清储奶罐中生鲜乳的耐热酶污染来源。多样性分析结果表明:牛舍垫土中的典型嗜冷菌丰富度,如假单胞菌属(1.57%)和不动杆菌属(1.56%)远高于其它样品,远高于头三把奶(0.26%,0.54%)。通过微生物产酶基因分析,储奶罐中生鲜乳中产生耐热酶的微生物主要来自嗜冷菌,为假单胞菌属和不动杆菌属。各样本中产蛋白酶的基因丰度为:牛舍垫土:8.71、头三把奶:0.18;产脂肪酶各的基因丰度为:牛舍垫土:16.19、TMR饲料:0.33、头三把奶:0.18、头三把奶之后生鲜乳:0.39、储奶罐中生鲜乳:0.22。此外,储奶罐中生鲜乳的脂肪酶基因在挤奶环节中基因重复率最低为57.14%。因此,微生物及其嗜冷菌通过牛舍垫土污染奶牛乳头,在挤奶环节会进入生鲜乳收集管路而造成污染。本研究为有效防控生鲜乳中耐热酶的污染提供了理论和数据基础。

Reactive molecular dynamics insight into the thermal decomposition mechanism of 2,6-Bis(picrylamino)-3,5-dinitropyridine

2,6-bis(picrylamino)-3,5-dinitropyridine(PYX)has excellent thermostability,which makes its thermal decomposition mechanism receive much attention.In this paper,the mechanism of PYX thermal decomposition was investigated thoroughly by the ReaxFF-lg force field combined with DFT-B3LYP(6-311++G)method.The detailed decomposition mechanism,small-molecule product evolution,and cluster evolution of PYX were mainly analyzed.In the initial stage of decomposition,the intramolecular hydrogen transfer reaction and the formation of dimerized clusters are earlier than the denitration reaction.With the progress of the reaction,one side of the bitter amino group is removed from the pyridine ring,and then the pyridine ring is cleaved.The final products produced in the thermal decomposition process are CO_(2),H_(2)O,N_(2),and H_(2).Among them,H_(2)O has the earliest generation time,and the reaction rate constant(k_(3))is the largest.Many clusters are formed during the decomposition of PYX,and the formation,aggregation,and decomposition of these clusters are strongly affected by temperature.At low temperatures(2500 K-2750 K),many clusters are formed.At high temperatures(2750 K-3250 K),the clusters aggregate to form larger clusters.At 3500 K,the large clusters decompose and become small.In the late stage of the reaction,H and N in the clusters escaped almost entirely,but more O was trapped in the clusters,which affected the auto-oxidation process of PYX.PYX's initial decomposition activation energy(E_(a))was calculated to be 126.58 kJ/mol.This work contributes to a theoretical understanding of PYX's entire thermal decomposition process.

基于计算设计的GH11家族木聚糖酶CDBFV的热稳定性改造及潜在机制研究

【目的】瘤胃真菌Neocallimastix patriciarum GH11家族木聚糖酶CDBFV在饲料、食品等领域具有良好应用前景,提高其热稳定性对其生产应用十分重要。【方法】使用分子动力学模拟、机器学习等策略设计潜在CDBFV热稳定性突变体,在大肠杆菌和毕赤酵母中进行异源表达纯化,测定最适反应条件、比酶活和85℃孵育3 min后相对剩余活力,通过结构分析明确热稳定性提高机制。【结果】位于CDBFV N端基序~(36)GNNS~(39)具有较高柔性,对其改造设计的单突变体N37P和N38V在85℃孵育3 min后,相对活力分别降低至70.3%和55.1%,较野生型(48.7%)提升了21.6%和6.5%;进而,在相对活力提升显著的N37P基础上叠加已报道优势突变体N88G,构建双突变体N37P/N88G,其相对活力达到了73.4%,较野生型提高了24.7%;此外,将N37P/N88G在毕赤酵母中进行了分泌表达,在85℃处理3 min后,相对活力达到了88.8%;结构分析表明,N37P突变的引入使得CDBFV形成了新的氢键相互作用,降低活性位点附近柔性,并干预了糖基化形成,进而提高了热稳定性。【结论】成功得到了高耐热双突变体N37P/N88G,为提高GH11家族木聚糖酶的热稳定性改造提供了新的思路和方法,有望推动CDBFV在饲料工业等高温环境下的广泛应用。