中温碱性脂肪酶的研究：V.扩展青霉PF868碱性脂肪酶在皮革及皮毛脱…

本文报导扩展青霉ＰＦ８６８产生的碱脂肪醇在动物皮革及皮毛脱脂工艺上的应用。试验结果表明：碱性脂肪酶２０￣３０ｕ／ｍＬ的浓度在ｐＨ９．０和温度３０￣３５℃条件下对猪皮、绵羊皮、兔皮及旱獭、狐狸和水貂皮毛具有良好的脱脂效果,脱脂率在８０％以上,高于传统碱法（Ｎａ２ＣＯ３）和表皮活性剂法（ＪＦＣ）。碱性脂肪酶不仅可脱尽皮板深层油脂,而且可以软化皮板。酶法脱脂的成革其抗张强度、伸长率、撕裂强度以及崩裂高度     

碱性脂肪酶与表面活性剂相互作用的研究

研究洗涤剂中常见的非离子表面活性剂、阴离子表面活性剂和阳离子表面活性剂对由扩展静霉（Ｐｅｎｉｃｉｌｌｉｕｍ ｅｘｐａｎｓｕｍ）产生碱性脂肪酶活性的影响及该酶在各种常见的洗涤剂溶液中的稳定性。     

Bohaisea-9145海洋低温碱性脂肪酶研究

从 2 0 0 0多份渤海海区海水海泥样品中分离获得一株新型脂肪酶高产菌株BohaiSea 914 5 ,经鉴定为适冷性海洋酵母 (Yarrowialipolytica)。菌株在以豆饼粉、棉籽饼粉和花生粕作为碳氮源并添加 0 5 %花生油的培养基中能较好地生长产酶 ,最适产酶温度 2 6± 1℃ ,产酶周期为 2 3h。所得脂肪酶的最适反应温度为 35℃ ,最适pH 8 5 ,pH4 0～ 9 0范围内稳定 ,热稳定性差。该酶与常见金属离子和化学试剂的配伍性较好 ,受表面活性剂SDS的激活 ,且具有良好的耐盐及抗氧化特性 ,是一种新型的海洋低温碱性脂肪酶 ,在洗涤剂行业特别是冷洗行业中具有良好的应用前景     

碱性脂肪酶的研究通过省级鉴定

湖南省微生物研究所研制的碱性脂肪酶于1988年12月在长沙通过省级鉴定。与会专家一致认为,碱性指肪酶应用于猪皮脱脂,效果好,优于现行使用的纯碱及脱脂剂,能提高皮质的品位,减少环境污染,具有明显的经济效益和社会效益。该酶产生菌扩展青霉CU-     

类产碱假单胞菌耐热碱性脂肪酶基因的克隆

将类产碱假单胞菌(Pseudomonas pseudoalcaligene)总DNA经Sau3AI部分酶解后的35～50kbDNA片段与经BamHI线性化及CIAP处理过的粘粒pIJ285连接,以大肠杆菌LE392为受体,构建类产碱假单胞菌的基因文库。通过三丁酸甘油酯平板和橄榄油平板法检测克隆子,获得一株具有耐热碱性脂肪酶活性的菌株LE392(pHZ1401)。随后将pHZ1401上的外源DNA片段进行亚克隆,从而获得了具有脂肪酶活性的菌株HB101(pHZ1402)和HB101(pHZ1403),它们分别携带有2.9kb和3.0kb的外源片段。两外源片段约有2kb的重叠区。HB101(pHZ1403)所分泌的脂肪酶活性比HB101(pHZ1402)高4倍,是出发菌的5倍。     

由扩展青霉产生碱性脂肪酶的纯化及其特性

国外报道了不少由细菌、霉菌、酶母菌产生的中性脂肪酶及由细菌产生的碱性脂肪酶的纯化方法,国内尚未见有关脂肪酶纯化的报道。为了进一步研究和扩大该酶在医药、食品等方面的应用范围,我们对由扩展青霉突变株UN-503产生的碱性脂肪酶进行了纯化及其特性的研究,现报道如下。     

紫外诱变原生质体选育低温碱性脂肪酶高产菌株

以产低温碱性脂肪酶约氏不动杆菌(Acinetobacter johnsonii)LP28为出发菌株,采用EDTA和溶菌酶处理制备原生质体.确定其最佳处理条件为37℃的水浴下,以终浓度为0.15 mg/mL的溶菌酶处理45 min,最终可获得90%的原生质体形成率及0.9%左右的再生率.采用紫外诱变原生质体的方法,筛选得...     

扩展青霉碱性脂肪酶基因在毕赤酵母中的高效表达

将编码扩展青霉碱性脂肪酶 (PEL)的cDNA克隆到酵母整合型质粒pPIC3.5K ,电转化His4缺陷型巴斯德毕赤酵母 (Pichiapastoris)GS115 ,通过橄榄油 MM平板及PCR方法筛选和鉴定重组子。重组子发酵液经SDS PAGE分析、橄榄油检验板鉴定 ,表明扩展青霉碱性脂肪酶基因在巴斯德毕赤酵母中获得了高效表达。表达蛋白分泌至培养基中 ,分子量约 2 8kD ,与扩展青霉碱性脂肪酶大小一致 ,占分泌蛋白的 95 %。橄榄油检验板检验表明该表达蛋白可分解橄榄油 ,通过优化该表达菌的发酵条件 ,以橄榄油为底物进行酶活测定 ,其发酵液酶活可达 2 6 0u mL。     

扩展青霉PF898碱性脂肪酶cDNA的克隆及序列分析

扩展青霉 (Penicilliumexpansum)PF898可产生一种具有工业价值的碱性脂肪酶 (PEL) .在测定了其N端 12个氨基酸残基序列的基础上 ,通过RT PCR、5′RACE、基因克隆及序列测定 ,获得了PEL完整的cDNA序列 (GenBank登录号为AF2 84 0 6 4 ) .cDNA全长 10 5 0bp ,包括PEL编码区、3′非翻译区和部分 5′非翻译区基因的序列 .编码区cDNA由 85 5个碱基组成 ,编码 1个由 2 85个氨基酸残基组成的酶蛋白 ,其信号肽及前肽部分由 2 7个氨基酸残基组成 ,成熟肽部分由 2 5 8个氨基酸残基组成 .根据氨基酸组成推导该脂肪酶蛋白的分子量为 2 7 3kD .该脂肪酶的氨基酸序列 130～ 134位上有各类脂肪酶中普遍存在的G X S X G保守序列     

类产碱假单胞菌耐热碱性脂肪酶的研究

从福建省福州市温泉澡堂污水浸润土壤中分离筛选到一株耐热碱性脂肪酶产生菌——类产碱假单胞菌(Pseudomonas pseudoalcaligenes)F331。产酶最适条件为:碳源小麦粉,氮源豆饼粉,起始培养pH9.4～9.5,培养温度24～26℃,培养周期32～34h。经硫酸铵盐析、Sepharose 4B和Sephadex G-200柱层析得到纯化的酶组分。该酶最适作用温度50℃,最适作用pH 10.0,60℃保温80min酶活基本不损失,在pH 7.0～10.0范围内酶蛋白稳定:Ca~(2+)和Mg~(2+)对酶有激活作用,Pb~(2+).Zn~(2+)、Fe~(2+)和Co~(2+)对酶活有抑制作用。该酶分子量45700。     

环氧交联剂和氨基载体固定化海洋假丝酵母脂肪酶*

游离酶经过固定化后,稳定性和环境耐受性得到提高,在食品、医药、化工、环境和皮革等领域可以很好的提高酶的利用率并降低生产成本,具有极大的应用潜力。新型交联剂在固定化酶工艺的应用极大推进了固定化酶研究的深入。借助新型交联剂聚乙二醇二缩水甘油醚(PEGDGE),利用氨基载体LX-1000HA固定化海洋假丝酵母脂肪酶,结合单因素和正交试验优化得到交联及固定化条件为:交联温度30℃,交联2h,交联剂浓度0.75%,pH7.0,加酶量800U,载体量0.5g,固定化2h,固定化温度45℃。根据上述最佳固定化工艺,制备得到固定化酶LX-1000HA-PEGDGE-CRL在最适条件下测得酶活达到160.81U/g,约为此前制备的固定化酶LX-1000HA-GA-CRL(由LX-1000HA和戊二醛交联脂肪酶得到)和LX-1000EA-PEGDGE-CRL(由短链氨基载体LX-1000EA和PEGDGE交联脂肪酶得到)酶活的2倍,发现固定化酶LX-1000HA-PEGDGE-CRL的最适反应温度相比于游离酶提高15℃;在70℃的环境中3h后酶活仍存留70%;循环使用6次后残留65%左右的酶活;酸碱耐受性和储存稳定性也表现良好,4℃保存30天后剩余约70%的初始酶活。同时,将制备的固定化酶LX-1000HA-PEGDGE-CRL与游离酶、固定化酶LX-1000HA-GA-CRL、固定化酶LX-1000EA-PEGDGE-CRL进行了比较,发现固定化酶LX-1000HA-PEGDGE-CRL在温度耐受性和重复使用性等方面具有更好的使用效果。     

Intracellular Lipase Formation by Washed Mycelium Biochemical Studies on Sclerotinia Libertiana. Part 13

Intracellular lipase of the fungus Sclerotina Libertiana Fcl. could be formed powerfully by washed mycelium during shaking in a plain buffer solution, just as well as in the case of shaking culture. Experiments showed revealed it to be favourable to set the mycelium in the experiment harvested at the end of its stationary phase of growth, and that the addition of various respiratory carbon sources had inhibiting effects, while several surface active agents and some enzyme preparations accelerating effects on the lipase formation. Also, the quality and the quantity of consumed cell-materials in the shaking experiment were investigated in relation to lipase formation.     

Characterization of Purified Staphylococcal Lipase

Purified staphylococcal lipase had an optimal pH of 8.3 for activity at 37 C, and an optimal temperature of 45 C at pH 8.0. During storage, the enzyme lost less than 10% of the activity over a period of 21 days at 4 and -23 C. The enzyme retained 93% of the activity when heated for 30 min at 50 C and was 95% destroyed in 30 min at 70 C. The purified lipase was capable of hydrolyzing a variety of natural fats and oils. However, the enzyme was three times more active on nonhydrogenated soybean oil than on hydrogenated soybean oil with an iodine value of <3.0. The enzyme was also capable of hydrolyzing fatty acids on the alpha, beta, and alpha' positions of a synthetic mixed triglyceride. In general, the presence of oxidizing agents increased the activity and the presence of reducing agents decreased the activity of the lipase enzyme.     

Enzymatic degreasing of a solid waste from the leather industry by lipases

The lipolytic activity of cutinase produced by a recombinant Saccharomyces cerevisiae strain was compared with Defat 50, a commercial lipase solution, for their degreasing abilities on a lipidic concentrate of sheep fleshing, a solid by-product of the leather industry. The specific activities obtained were 2749 and 938 U mg^–1 of lipase for cutinase and Defat 50, respectively.     

A model of evaporative cooling of wet skin surface and fur layer

(1) A theoretical model that simultaneously solves heat and mass transfer in a wet skin surface and fur layer that occurs when an animal is cooled by blowing air over its wetted skin surface and hair coat is presented. (2) The model predicts evaporative and convective heat losses for different levels of wetness, air velocity, ambient temperature, relative humidity and fur properties. (3) Model predictions provide insight about evaporative and convective cooling processes of cows in stressful hot environments.     

Characterization of a New Epidemic Necrotic Pyoderma in Fur Animals and Its Association with Arcanobacterium phocae Infection

A new type of pyoderma was detected in Finnish fur animals in 2007. The disease continues to spread within and between farms, with severe and potentially fatal symptoms. It compromises animal welfare and causes considerable economic losses to farmers. A case-control study was performed in 2010–2011 to describe the entity and to identify the causative agent. Altogether 99 fur animals were necropsied followed by pathological and microbiological examination. The data indicated that the disease clinically manifests in mink (Neovison vison) by necrotic dermatitis of the feet and facial skin. In finnraccoons (Nyctereutes procyonoides), it causes painful abscesses in the paws. Foxes (Vulpes lagopus) are affected by severe conjunctivitis and the infection rapidly spreads to the eyelids and facial skin. A common finding at necropsy was necrotic pyoderma. Microbiological analysis revealed the presence of a number of potential causative agents, including a novel Streptococcus sp. The common finding from all diseased animals of all species was Arcanobacterium phocae. This bacterium has previously been isolated from marine mammals with skin lesions but this is the first report of A. phocae isolated in fur animals with pyoderma. The results obtained from this study implicate A. phocae as a potential causative pathogen of fur animal epidemic necrotic pyoderma (FENP) and support observations that the epidemic may have originated in a species -shift of the causative agent from marine mammals. The variable disease pattern and the presence of other infectious agents (in particular the novel Streptococcus sp.) suggest a multifactorial etiology for FENP, and further studies are needed to determine the environmental, immunological and infectious factors contributing to the disease.     

微环境对脂肪酶催化拆分外消旋2－辛醇的影响

微环境对脂肪酶催化拆分外消旋２－辛醇的影响　　　　　　　杨红,曹淑桂,韩四平,黄仲丽,杨同书（吉林大学酶工程国家重点实验室,长春１３００２３）手性２－辛醇不仅是制备液晶材料不可缺少的重要手性原料,也是合成具有光学活性的医药和农药的重要手性中间体．本文...     

Expression and Biochemical Characterization of a Thermophilic Organic Solvent-Tolerant Lipase from Bacillus sp. DR90

The objective of the present study was the isolation, molecular cloning and biochemical characterization of a thermophilic organic solvent-resistant lipase from Bacillus sp. DR90. The lipase gene was expressed in Escherichia coli BL21(DE3) using pET-28a(+) vector. The purification of recombinant lipase was conducted by nickel affinity chromatography and its biochemical properties were determined. The lipase sequence with an ORF of 639 bp contains the conserved pentapeptide Ala-His-Ser-Met-Gly. His-tagged recombinant lipase had a specific activity of 1,126 U/mg with a molecular mass of 26.8 kDa. The cloned lipase was optimally active at pH 8.0 and 75 °C representing high stability in broad ranges of temperature and pH. High performance liquid chromatography was used to determine the major compounds released during the lipase-catalyzed reaction of p-nitrophenyl derivatives as well as the substrate specificity. The purified lipase showed high compatibility towards various organic solvents, surfactants and commercial solid/liquid detergents; therefore the recombinant DR90 lipase could be considered as a probable candidate for future applications, predominantly in detergent processing industries.     

The Effect of Ca and Antidiuretic Hormone on Na Transport across Frog Skin : II. Sites and mechanisms of action

A method has been developed for determining unidirectional Na fluxes across the two faces of the transporting cells in the frog skin. The method has been used to investigate the location of the sites at which Ca and anti-diuretic hormone act to alter the rate of active Na transport across the skin. The results have indicated that the primary effect of both agents is on the Na permeability of the outward facing membrane of the cells. Ca decreases and the hormone increases permeability of this barrier. Neither agent appears to have a direct effect on the active transport system itself assuming that it is located at the inner membrane of the cells. The rate of active Na transport is altered as a result of changes in the size of the Na pool in the cells which occur because of changes in the rate of Na entry through the outer membrane. Thus, the results indicate that the Na permeability of the outer membrane plays an important role in controlling the rate of net active Na transport across the skin.     

Lipase of Mucor pusillus

Lipase of Mucor pusillus NRRL 2543 was recovered with ammonium sulfate precipitation, gel filtration on Sephadex G-75, and anion-exchange chromatography on diethylaminoethyl-Sephadex A-50. Maximal glycerol ester hydrolase (lipase) activity was observed at pH 5.0 to 5.5 and 50 C when trioctanoin and olive oil were used as substrates. The enzyme also showed esterase activity; it hydrolyzed, with the exception of methyl butyrate, all methyl esters tested. A minimum chain length of six carbons appeared to be a requirement for esterase activity, which was maximal at about pH 5.5 with methyl dodecanoate (C(12)) as the substrate. Neither the glycerol ester hydrolase (lipase) nor the esterase activity of the enzyme appeared to be affected by thiol group inhibitors, chelating agents, and reducing compounds. On the other hand, hydrolysis of triolein and methyl dodecanoate was arrested to the same extent in the presence of diisopropyl fluorophosphate, which suggested the involvement of serine in the active center of the enzyme. The enzyme remained stable during a 30-day storage at - 10 C.