嗜热菌Anoxybacillus sp．DL3产蛋白酶条件及其酶学性质研究

目的：对Anoxybacillussp．DL3的产蛋白酶条件及其酶学性质进行研究,为下一步进行蛋白酶基因的克隆、表达提供依据。方法：应用常规方法液体培养细菌,研究温度、pH、培养基中碳源、氮源对菌株产蛋白酶的影响,硫酸铵盐析的方法提取酶液,并采用Folin法测酶活性。用紫外分光光度计在OD680hi／1测吸光值。并对提取的蛋白酶液进行酶的最适温度、pH以及酶的热稳定性和pH稳定性研究,向酶液中添加金属离子和EDTA、PMSF,研究其对酶活性的影响。结果：在培养基初始pH是6．5,培养温度为40℃时菌株产酶酶活性最大;培养基中以乳糖为碳源,酵母膏和硫酸铵为氮源,碳源与氮源的比例为1：2时,酶活最大。酶学性质研究结果显示：该酶的最适反应温度是55℃,最适反应pH是7．0;在50℃保温20min-80min内,酶活力下降幅度较小。60℃保温60min后,仍保持约60％的酶活。70℃保温60min后,残余酶活为30％。该酶在pH为6．0～8．0范围内,相对酶活差别不是很大,下降趋势大致相同。在强碱条件下,相对酶活下降很明显。Fe2＋、Cu2＋和Hg2＋对酶活性有明显的抑制作用;Ca2＋、Mg^2＋、Mn2＋等金属离子对酶活性有明显的促进作用;乙二胺四乙酸（EDTA）和苯甲基磺酰氟（PMSF）对酶活性也有一定抑制作用。结论：Anoxybacillussp．DL3所产的蛋白酶为嗜热中性蛋白酶,此酶具有较好的热稳定性和pH耐受性,该菌株具有进一步开发、利用的价值。     

Aspergillus sp.脂肪酶发酵条件优化及酶学性质的研究

作者为了得到一种热稳定性较好的脂肪酶新酶种,通过研究分离白极端环境的Aspergillus sp．的最佳产酶条件及其所产脂肪酶的酶学性质,得出了该菌产酶的最佳发酵条件为：以1％黄豆饼粉为氮源、0．2％玉米淀粉为碳源,1．5％橄榄油为诱导物,起始pH6．0左右。装量10mL（250mL三角瓶。摇瓶转速180r／min）、发酵时间为96h。在最佳发酵条件下可得最大发酵酶活36U/mL。Aspergillus sp．所产的脂肪酶的酶学性质是：最适pH为9．0,在pH5．0—10．0于20℃下放置24h后,残余酶活仍保持在起始酶活的90％以上;该酶的最适温度为50℃,50℃保温60min后仍保留70％以上的酶活。Aspergillus sp．所产脂肪酶的热稳定性较好。     

白地霉Cryytococcus neoformans脂肪酶的双水相萃取

本文研究了不同无机盐的双水相体系对白地霉脂肪酶的萃取分离效果,对PEG/（NH4）2SO4成相系统进行了系统的研究,通过考察体系PEG分子量、不同的无机盐、PEG浓度、（NH4）2SO4浓度、离子强度、pH值及（NH4）2SO4浓度对反萃取的影响,并通过正交实验进一步优化了实验条件,初步确定在PEG浓度15%,（NH4）2SO4浓度22.5%,pH8.0,不加NaCl的条件下进行双水相萃取,脂肪酶分离系数和纯化倍数分别为6.8和7.5,比活力达到40.3 U/mg蛋白。     

生淀粉糖化酶产生菌Cellulosimicrobium sp.SDE的分离鉴定及酶学性质研究

从淀粉制品厂周围土壤中分离到一株高产生淀粉糖化酶的菌株SDE,经形态、生理生化及16S rDNA序列分析将其鉴定为Cellulosimicrobium sp..SDE菌株的最适产酶条件为pH7.0,培养温度为30℃.培养42h粗酶液的酶活达175.3U/mL.该酶以生玉米淀粉为底物时,最适作用pH6.0,最适作用温度40℃,pH6.0-7.0范围内酶活力稳定.在Ca2 存在下,酶的热稳定性很高,80℃保温1 h后,酶活力仍保持50%.Ba2 、Cu2 对酶活有强烈的抑制作用,Ca2 、Zn2 有很强的激活作用.     

低温脂肪酶的产酶条件优化及其酶学性质

利用单因素筛选和正交试验对Burkholderia sp. SYBC LIP-Y发酵产酶的液体培养基和发酵条件进行了优化,其优化配方为：可溶性淀粉10 g/L、牛肉膏15 g/L、NaNO3 0.252 g/L、橄榄油40ml/L、Triton x-100 10ml/L、初始pH 7.5、接种量10%(V/V),脂肪酶酶活达到85.23U/ml,是优化前的3.63倍。通过对双水相纯化得到的脂肪酶进行酶学性质研究,确定该酶反应的最适pH为10.0,最适温度为30℃,40℃下保温60min酶活性还有80%以上,该脂肪酶为低温脂肪酶,热稳定性好,具有一定的耐醇性,应用前景广阔。     

Pseudomonas sp. RT-1低温脂肪酶的纯化及酶学特性

Pseudomonas sp. RT-1是从低温环境下分离的低温脂肪酶产生菌,对该菌产生的胞外脂肪酶(PL-1)进行纯化,并对其酶学特性进行初步研究。Pseudomonas sp. RT-1的发酵上清液经60%(NH4)2SO4沉淀、12~14000截留相对分子质量(MWCO)透析袋透析、Sephadex G75分子筛和超滤浓缩后,得到了电泳纯的P-L1。SDS-PAGE电泳估算其表观相对分子质量为4.43×104。对其酶学特性研究表明:PL-1是低温碱性脂肪酶且对有机溶剂的耐受性较好。10~40℃内有较好的催化活性,最适作用温度为18℃;0~50℃该酶的稳定性较好,当温度超过50℃时则容易失活;最适作用pH为10.2,且pH在9~11时较稳定;该酶对有机溶剂的耐受性较好,10mmol/L的Ca2+、K+、Na+和Fe3+对PL1的酶活力有促进作用,其中Ca2+促进作用最大,提高了146.07%,而10mmol/L的Cu2+、Co2+、Mn2+、Mg2+、Zn2+、Ba2+和Al3+对酶活力具有不同程度的抑制作用,其中Al3+抑制作用最强,抑制了98.55%;PL-1对C链长度小于或等于12的短链脂肪酸形成的甘油三酯具有较强的水解能力;1mmol/L的去氧胆酸盐(desoxycholate)和0.01%的Triton X100对酶活力具有提高作用,分别提高了30.74%和11.83%;0.01%的SDS和Tween-80、1mmol/L的EDTA和尿素对酶活都有抑制作用,其中EDTA的抑制作用最大,抑制了80%。     

产低温脂肪酶菌株Psychrobacter sp.7342的筛选及粗酶性质研究

从南北极环境土样中筛选到1株产脂肪酶细菌7342,16SrDNA序列分析表明该菌株属于Psychrobacter sp..p-NPP法研究显示,菌株7342所产粗酶液的最适温度为30℃、最适pH值为8.0,对热较稳定;Co2+和Cs+对粗酶液有激活作用,而Na+、Sr2+等7种金属离子对其均有不同程度的抑制作用;粗酶液能在高浓度的SDS、Tween20等变性剂中表现出较好的稳定性.     

海洋来源琼胶酶的分离纯化及酶学性质研究

采用Vibiro sp.ZC-1发酵制备琼胶酶,粗酶液经过中空纤维柱浓缩、硫酸铵沉淀、DEAE-阴离子交换层析,得到一个电泳纯的琼胶酶组分Aga ZC-1,其分子质量约为45k Da,比活力为114.613U/mg。对Aga ZC-1进行酶学性质分析,结果表明,其最适反应p H为7.0,在p H为5.0~9.0时保温1h仍能保持80%以上的酶活力;最适反应温度为50℃,在45℃条件下保温1h酶活力保持在60%以上。在高浓度(5mmol/L)下,Fe~(3+)、Cu~(2+)、Sn~(2+)和Zn~(2+)能完全抑制琼胶酶的活性,在低浓度(1mmol/L)下,Cu~(2+)、Ba~(2+)、Na~+、Zn~(2+)、Ag~+、Sr~(3+)、K+对琼胶酶活性具有明显抑制作用。琼胶酶的动力学参数K_m和V_(max)分别为0.538mg/ml和6.33μmol/(L·min),对琼胶底物具有高度专一性,降解产物主要为新琼四糖和新琼六糖。     

丝孢酵母脂肪酶的酶学性质和化学修饰

实验室筛选到的一株丝孢酵母Trichosporon sp.脂肪酶经过硫酸铵沉淀和一系列的层析步骤分离纯化到电泳纯。对纯酶的酶学性质研究表明,此酶的分子量为28kD,pI为pH8.7,最适作用温度40℃,最适作用pH为8.0。随后利用不同的化学修饰剂对酶蛋白进行修饰,通过酶催化活力的改变对位于酶活性位的氨基酸残基进行分析,结果表明酶活性位可能含有组氨酸、丝氨酸和谷氨酸(或天冬氨酸)。最后,对此酶的氨基酸成分进行了分析,结果表明,此酶分子中天冬氨酸、丝氨酸、谷氨酸、甘氨酸、丙氨酸含量高,两种碱性氨基酸精氨酸、赖氨酸及组氨酸的含量也比较高,而脯氨酸、色氨酸的含量相对较低。     

极端环境脂肪酶菌种库建立及其酶学性质研究

为得到特殊性质的脂肪酶酶种,从来源于极端环境的27份样品定向筛选脂肪酶产生菌,并构建了一个小型的菌种库。其中酵母L112在20℃,pH5．0时酶活为18．60u/ml,属低温酸性酶种。酶学性质研究表明,该酶最适作用温度为25-30℃,最适pH为5.4。在pH4.8,50℃条件下40min保持60%以上酶活。Mg^2 和Ca^2 对酶有激活作用,Zn^2 、Fe^2 、Cu^2 和EDTA有抑制作用。该产酶菌析经中国科学院微生物研究所鉴定为罗伦隐球酵母（Cryptococcus laurentii(Kufferath)C.E.Slcinner).     

Purification and partial characterization of two new cold-adapted lipases from mesophilic Geotrichum sp. SYBC WU-3

Two cold-adapted lipases (Lipase-A and Lipase-B in the paper) of mesophilic Geotrichum sp. SYBC WU-3 were purified by using (NH₄)₂SO₄ fractionation, chromatography separation on a DEAE-cellulose-32 column and a Sephadex G100 column. The molecular mass of Lipase-A and Lipase-B were determined to be approximately 41.1 and 35.8 kDa, respectively by SDS-PAGE. The optimum temperature for the activity of Lipase-A was found to be 20 °C, and that of Lipase-B was 15 °C. Lipase-A and Lipase-B had good stability when temperature was below 40 °C. Both the optimum pH for the activity of the lipases was 9.5. Lipase-A retained about 80% of its activity when pH was between 3 and 6 and Lipase-B maintained over 80% activity in the pH range of 3–8. The two lipases showed hydrolysis efficiency to various p-nitrophenyl esters, but they were more active with shorter p-nitrophenyl esters (C2 and C4).     

Nematicidal metabolites produced by the endophytic fungus Geotrichum sp. AL4

From the endophytic fungal strain Geotrichum sp. AL4, cultivated from the leaves of the neem tree (Azadirachta indica), four compounds, 1-4, were isolated from the AcOEt extract, including two new, chlorinated, epimeric 1,3-oxazinane derivatives. All compounds were assessed for their nematicidal activities against the nematodes Bursaphelenchus xylophilus and Panagrellus redivivus, and three out of the four isolates showed noticeable bioactivities.     

白地霉ch-3脂肪酶Ⅰ基因在大肠杆菌中的表达

构建了白地霉脂肪酶Ⅰ的基因工程菌,为进一步进行蛋白质工程改造和脂肪酶应用奠定了基础。从新疆昌吉市油脂化工厂含油冻土中分离得到1株低温脂肪酶产生菌-白地霉ch-3。该菌发酵上清液中的脂肪酶最适作用温度为35℃,在0℃仍可保持66%的相对酶活力。应用PCR技术从白地霉ch-3基因组DNA中克隆得到脂肪酶Ⅰ基因lip1,将该基因与原核表达质粒载体pET-22b（＋）连接,构建重组质粒pETl-ip1,转化E.coliBL21（DE3）,酶切鉴定,筛选得到重组菌。十二烷基磺酸钠-聚丙希酰胺（SDS-PAGE）显示重组脂肪酶Ⅰ的相对分子质量约为5.8×10^4,酶活为2.73 U/mL,表明lip1基因的表达产物具有正常的生物学活性。白地霉ch-3脂肪酶Ⅰ基因lip1能够在大肠杆菌中有效地表达。     

Purification and characterization of a mesophilic organic solvent tolerant lipase produced by Acinetobacter sp. K5b4

The extracellular lipase produced by Acinetobacter sp. K5b4 was purified to homogeneity using ultrafiltration (cutoff 30?KDa) followed by gel filtration chromatography on Sephadex G-50. The enzyme was purified to homogeneity with an apparent molecular mass of 133?KDa by SDS-PAGE. This purification resulted on 10.24 fold with 18.3% recovery. The K_m and V_max of purified enzyme when using pNPL hydrolysis were 4.0?mM and 73.53?nmol/ml/min, respectively. The pure enzyme was greatly stimulated in the presence of 20, 40 and 60% (v/v) methanol, DMSO and acetone whereas, ethanol, acetonitrile and propanol decreased the enzyme activity. Maximum enzyme activity was achieved at pH 7.0 and incubation temperature of 27?°C. The enzyme was stable within a pH range of 6.5 to 7 at 27?°C for 1?h. The enzyme activity was enhanced up to 36% by KCl, BaCl₂, MgCl₂ and CaCl₂ while obviously inhibited (10–20%) by CoCl₂, ZnCl₂, MnCl₂ and CuCl₂. No inhibitory effects were observed with 1.0 and 5.0?mM of 2-mercaptoethanol and EDTA. Similarly, SDS at 1.0?mM does not affect the enzyme activity while high reduction (80%) was observed at 5.0?mM SDS concentration. The enzyme was active against p-nitrophenyl esters of C8, C12 and C16 with highest preference to the medium carbon chain p-nitrophenyl caprylate (C8). The fact that the enzyme displays distinct stability in the presence of methanol, DMSO and acetone suggests that this lipase is suitable as biocatalyst in organic synthesis where such hydrophilic organic solvents are used as a reaction media.     

Purification and characterization of an exopolygalacturonase produced by Fusarium oxysporum f. sp. radicis lycopersici

Abstract An exopolygalacturonase produced by Fusarium oxysporum f. sp. radicis lycopersici , a fungus that produces root rot, was purified by gel filtration and ion exchange chromatography. It had a M _r 68 K, a pH optimum of 5.6 and an optimum temperature of 60°C. This polygalacturonase was inhibited by calcium ions and had a K _m of 0.64 mM using sodium polypectate as substrate. The exo mode of action of this enzyme was revealed by thin-layer chromatography of hydrolysed substrate.     

Production, purification and characterization of chitosanase produced by Gongronella sp. JG

AIMS: To optimize the production condition of chitosanases of Gongronella sp. JG and to characterize the major chitosanase. METHODS AND RESULTS: In the optimized medium and culturing condition, strain JG produced 800 micromol min(-1) l(-1) chitosanase activity at 72 h. The major chitosanase - csn1 was purified through three chromatography steps: CM (carboxymethyl)-Sepharose fast flow (FF), Sephacryl S200, SP (sulfopropyl)-Sepharose FF. The molecular weight and the pI value of csn1 were about 90,000 Da and 5 x 8, respectively. Its specific activity was 82 micromol min(-1) mg(-1). The optimal reaction pH for csn1 was between 4 x 6 and 4 x 8. The optimal reaction temperature was 50 degrees C. The half-life of csn1 at 50 degrees C was estimated to be about 65 min. Mn(2+) was a strong stimulator of csn1 activity, both at 1 and 10 mmol l(-1). csn1 showed its highest activity with chitosan of 85% degree of deacetylation, but did not hydrolyse colloidal chitin and carboxylmethyl cellulose. In 20 mmol l(-1) sodium acetate buffer (pH 4 x 8) and at 50 degrees C, the K(m) of csn1 was calculated to be 4 x 5 mg ml(-1). CONCLUSIONS: The production condition of chitosanases by Gongronella JG was optimized and the major chitosanase, csn1, was characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: The present work for the first time reported the production, purification and characterization of chitosanases produced by fungus of Gongronella sp. These results provided us more information on fungal chitosanases.     

Different bioreactor configurations for the decolourisation of the azo dye reactive black 5 by Geotrichum sp. CCMI 1019

Decolourisation of the azo dye Reactive Black 5 by Geotrichum sp. CCMI 1019 was studied using stirred tank reactors (STR) and two types of bubble columns (porous plate (PP) bubble column and aeration tube (AT) bubble column). For the bubble columns, the k_La increased with the gas fractional hold-up (ε_G) and the aeration rate. A linear relationship between ε_G and superficial gas velocity was obtained for all reactors. At same aeration rates, the PP bubble columns showed higher k_La and hold-up values than the AT bubble column. In the STRs, large and dense aggregates were formed which adhered to surfaces whereas bubble columns gave smaller and less compact pellets.

Manganese peroxidase and laccase were detected in the extracellular media in all reactors. However, laccase was only detected after the onset of decolourisation, suggesting that additional enzymes may be involved. Mn peroxidase activity was detected (about 46 U/ml) in both the STRs and AT bubble columns but higher values (110 U/ml) were obtained with the PP bubble columns.

Out of the three reactor systems studied, the AT bubble columns gave the most favourable results for Reactive Black 5 decolourisation. Rapid and complete colour removal was obtained throughout the visible spectrum. Bubble columns are simple in design as well as operation and may be useful for the bioremediation of textile wastewater.     

Different bioreactor configurations for the decolourisation of the azo dye reactive black 5 by Geotrichum sp. CCMI 1019

Decolourisation of the azo dye Reactive Black 5 by Geotrichum sp. CCMI 1019 was studied using stirred tank reactors (STR) and two types of bubble columns (porous plate (PP) bubble column and aeration tube (AT) bubble column). For the bubble columns, the k_La increased with the gas fractional hold-up (ε_G) and the aeration rate. A linear relationship between ε_G and superficial gas velocity was obtained for all reactors. At same aeration rates, the PP bubble columns showed higher k_La and hold-up values than the AT bubble column. In the STRs, large and dense aggregates were formed which adhered to surfaces whereas bubble columns gave smaller and less compact pellets.

Manganese peroxidase and laccase were detected in the extracellular media in all reactors. However, laccase was only detected after the onset of decolourisation, suggesting that additional enzymes may be involved. Mn peroxidase activity was detected (about 46 U/ml) in both the STRs and AT bubble columns but higher values (110 U/ml) were obtained with the PP bubble columns.

Out of the three reactor systems studied, the AT bubble columns gave the most favourable results for Reactive Black 5 decolourisation. Rapid and complete colour removal was obtained throughout the visible spectrum. Bubble columns are simple in design as well as operation and may be useful for the bioremediation of textile wastewater.     

链霉菌G4溶菌酶的产生条件及特性

对链霉菌G4的产酶发酵条件和溶菌特性进行研究结果表明:蔗糖30 g/L、大豆蛋白胨12.5 g/L、牛肉膏2 g/L,对产酶最为有利;G4溶菌酶最适培养温度33 ℃,培养时间72 h,培养基初始pH 8.G4溶菌酶的最适作用温度和最适作用pH分别是55 ℃和6.5,多数金属离子会抑制G4溶菌酶的活性,其中Zn2+、Cu2+、Fe2+、 Pb2+几乎可以使其完全失活;对几种细菌、酵母菌的研究表明,G4溶菌酶对卵清溶菌酶不能作用的变形链球菌和金黄色葡萄球菌有很强的溶解活性.