An extra-cellular alkaline metallolipase from Bacillus licheniformisMTCC 6824: Purification and biochemical characterization

An extra-cellular lipase produced by Bacillus licheniformis MTCC 6824 was purified to homogeneity by ammonium sulphate fractionation, ethanol/ether precipitation, dialysis, followed by anion-exchange chromatography on Amberlite IRA 410 (Cl⁻ form) and gel exclusion chromatography on Sephadex G 100 using Tris–HCl buffer (pH 8.0). The crude lipase extract had an activity of 41.7 LU/ml of culture medium when the bacterium was cultured for 48 h at 37 °C and pH 8.0 with nutrient broth supplemented with sardine oil as carbon source. The enzyme was purified 208-fold with 8.36% recovery and a specific activity of 520 LU/mg after gel exclusion chromatography. The pure enzyme is a monomeric protein and has an apparent molecular mass of 74.8 kDa. The lipase had a V_max and K_m of 0.64 mM/mg/min and 29 mM, respectively, with 4-nitro phenylpalmitate as a substrate, as calculated from the Lineweaver–Burk plot. The lipase exhibited optimum activity at 45 °C and pH 8.0, respectively. The enzyme had half-lives (T_1/2) of 82 min at 45 °C, and 48 min at 55 °C. The catalytic activity was enhanced by Ca²⁺ (18%) and Mg²⁺ (12%) at 30 mM. The lipase was inhibited by Co²⁺, Cu²⁺, Zn²⁺, Fe² even at low concentration (10 mM). EDTA, at 70 mM concentration, significantly inhibited the activity of lipase. Phenyl methyl sulfonyl fluoride (PMSF, 70 mM) completely inactivated the original lipase. A combination of Ca²⁺ and sorbitol induced a synergistic effect on the activity of lipase with a significantly high residual activity (100%), even after 45 min, as compared to 91.5% when incubated with Ca²⁺ alone. The lipase was found to be hydrolytically resistant toward triacylglycerols with more double bonds.     

Production of n-3 polyunsaturated fatty acid concentrate from sardine oil by immobilized Candida rugosa lipase

ABSTRACT:  This study was conducted to develop an immobilized-enzyme system to entrap lipase in chitosan-alginate-CaCl₂ beads for the purpose of concentrating n-3 polyunsaturated fatty acids (n-3 PUFAs) from sardine oil. Lipase was immobilized by an ionotropic gelatin method analyzed for characteristics. Optimum pH of immobilized lipase shifted from pH 7.0 to 6.0 and immobilized lipase showed higher stability against pH and temperature changes. Original sardine oil contained 38.1% n-3 PUFAs (25.2% 20:5n3 and 7.20% 22:6n3), and the concentration was significantly increased to 65.3% (40.2% 20:5n3 and 15.5% 22:6n3) with free lipase and to 64.8% (39.6% 20:5n3 and 15.3% 22:6n3) with immobilized lipase after 90 min of repeated hydrolysis. Fatty acid content of the free fatty acid (FFA) fraction of hydrolyzed oil showed that lipase preferably hydrolyzed 16:0, 16:1n7 and 18:0 accounting for 76.6% and 69.5% of total FFAs (after 1st and 2nd hydrolysis, respectively). This study shows that use of immobilized lipase systems for increasing n-3 PUFA concentration in sardine oil provides new processing opportunities for the industry.     

麦胚稳定化处理方法的比较研究

研究了热风干燥、微波干燥、常压蒸气和高压蒸气处理麦胚对其加速储藏期间脂肪酶的钝化效果.在38℃下进行35 d的加速储藏试验,并跟踪检测,以酸值和过氧化值作为钝化脂肪酶的表征参数,对不同方法处理原料的优化结果(酸值和过氧化值)进行了比较.结果表明,脂肪酶的钝化效果:湿热蒸气处理的效果优于微波干燥、明显好于热风干燥处理,酸值和过氧化值均上升缓慢,常压蒸气处理20 min即可达到预定效果.对常压蒸气处理的麦胚原料进行超临界萃取并对得到的麦胚油进行了GC分析,与对照组原料相比,脂肪酸组成与含量均无明显变化.水蒸气是良好的热载体和传热介质,蒸气处理能够有效钝化脂肪酶的活力,提高小麦胚的贮藏稳定性.     

微波辐照稳定化处理小麦籽粒及其品质变化的研究

以河北优质白麦为原料,研究了微波辐照功率、辐照时间、润麦水分、润麦时间等参数对全麦粉脂肪酶活动度、湿面筋含量的影响,考察了适度微波辐照对面粉粉质特性和糊化特性的影响。结果表明微波辐照能够显著降低(p<0.05)全麦粉的脂肪酶活动度和湿面筋含量。适度的微波辐照(微波功率420 W,辐照时间90 s,润麦水分14%,润麦时间25 min)能够大幅降低全麦粉脂肪酶活动度,同时对其湿面筋含量损伤较小,并提高面粉粉质稳定时间,降低弱化度,增加了面糊峰值粘度和回生值,改善了面筋强度和面糊稳定性,延缓了全麦粉脂肪酸值的升高。     

米糠毛油酶法脱酸的工艺优化

米糠毛油普遍酸价较高,采用常规的脱酸方式存在炼耗高、预处理要求高、营养成分损失大、油品品质差等问题。酶法脱酸可以有效避免上述问题。采用固定化脂肪酶CALB对米糠毛油进行酶法脱酸研究,得到优化工艺条件为甘油与游离脂肪酸摩尔比1∶3、剪切速度25 m/s、剪切时间20min、加酶量50 g/kg、反应温度70℃、反应时间6 h、甘油中水分含量25%、真空度小于1 000 Pa、通氮气,在此条件下酯化率可达92. 5%。     

新型液态固定化脂肪酶制备及其稳定性的研究

传统的固态固定化脂肪酶存在载体易破碎、制备成本高等问题,制约了其工业应用。利用前期开发的三液相体系中相作为脂肪酶液态固定化载体,研究了不同种类的成相物质、成相物质的比例以及不同种类的脂肪酶对液态固定化酶回收率的影响。同时考察了该液态固定化载体固定脂肪酶的稳定性。结果表明:正己烷/PEG400/Na_2SO_4三液相体系中相为更优的液态固定化酶载体;最优的制备条件为PEG400比例20%、Na_2SO_4比例14%,此条件下AY30脂肪酶的回收率可达98%以上,其他类型脂肪酶中相回收率均大于90%;该液态固定化载体在30℃的环境下能有效保持脂肪酶的催化活性,保护效果在不同温度下均明显优于传统水-有机溶剂体系;同时,脂肪酶在此液态固定化载体中的保藏稳定性也优于水环境,低温保藏2周后仍能保留约78. 4%的酶活力,体现出较好的保藏稳定性。     

聚合物接枝脂肪酶的合成及其对酶活的影响

利用原子转移自由基聚合方法将含C₂～C₄烷基链的单体化合物分别接枝到皱褶假丝酵母脂肪酶（CRL）表面合成了HEMA-g-CRL、GMA-g-CRL、nPMA-g-CRL和BMA-g-CRL四种聚合物接枝CRL。CD和荧光光谱学分析显示,聚合物接枝导致HEMA-g-CRL、nPMA-g-CRL和BMA-g-CRL分子的α-螺旋和β-折叠含量增加。与此同时,聚合物接枝CRL分子荧光发射光谱也发生了蓝移,意味着其具有更加紧密的分子构象。酶活测定结果进一步显示,聚合物接枝增强CRL酶活227%～278%。随着单体化合物烷基链长增加,聚合物接枝CRL的K _m值由0.17 mmol·L^-1降至0.09 mmol·L^-1。与此同时,聚合物接枝CRL的k _cat值则提高至106～182 s^-1。BMA-g-CRL的催化效率达到了野生型CRL的3.28倍。这反映出聚合物接枝助力了CRL“盖子”结构开启和CRL活性中心暴露,促进底物的转化。稳定性测试表明,聚合物接枝提升了CRL的热稳定性并拓宽了其pH操作范围。     

除膻菌株分离鉴定及其对膻味脂肪酸降解能力的研究

为了分离筛选出能作用于膻味脂肪酸的微生物,从火腿和土壤中分离出11株菌,这11株菌均能对羊油脂进行水解。通过形态学、生理生化特征鉴定及16S rRNA基因序列和ITS基因序列分子生物学鉴定,确定为6株细菌和5株霉菌。分别将11株菌接种到加入4-甲基辛酸和4-乙基辛酸的液体培养基进行发酵,发现4-甲基辛酸的减少量达到30%～70%,4-乙基辛酸减少量达到10%～70%。这11株菌对膻味脂肪酸具有不同程度的降解作用,该研究结果可为微生物羊肉除膻提供理论基础。