Purification and characterization of hepatic triacylglycerol lipase isolated from rainbow trout,Oncorhynchus mykiss

Trialcylglycerol (TG) lipase was isolated and partially purified from rainbow trout liver. Triacylglycerol lipase activity was assayed by measuring¹⁴C-oleic acid release from¹⁴C-triolein.¹⁴C-oleic acid release was linear for up to two hours. Optimal activity occurred at pH 7.0 and 15°C. Most of the lipase activity was recovered in the cytosolic fraction. A 27,000-fold purification was achieved after Sepharose (Bio-gel A 0.5 M, 200–400 mesh) chromatography of a resuspended 20% ammonium sulfate fraction. The molecular weight of the trout hepatic lipase as determined by size-exclusion chromatography and by SDS-polyacrylamide gel electrophoresis was 40–43 kD. Lipase-mediated hydrolysis of TG resulted in the production of diacylglycerols, monoacylglycerols, and fatty acids. Kinetic analysis indicated that V_max=0.016 nmol/h/mg protein and that K_m=0.28 mM triolein. Lipolytic activity was enhanced in the presence of cAMP/ATP-Mg²⁺. These results suggest that the liver of trout possesses a neutral TG lipase that is responsible for mobilizing stored TG and is catalytically activated by phosphorylation.A part of this work was presented at the Annual Meeting of the American Society of Zoologists, December 26–30, 1990, San Antonio, TX.     

罗非鱼鱼皮提取明胶的酶法脱脂工艺研究

用脂肪酶对罗非鱼鱼皮进行酶法脱脂工艺研究。通过单因素与正交试验确定最适的酶法脱脂工艺为:pH值9,脱脂时间3h,酶添加量2%,料液比1:4,温度40℃,该工艺的脱脂率可达67.5%。通过超声波辅助脱脂研究表明其脱脂率并无明显的提高。     

家蚕对BmNPV抗病性与中肠消化酶活性的关系

对6个现行家蚕品种中肠消化酶活性与家蚕对BmNPV病毒抗性比较与分析,以期寻找现行家蚕品种消化酶的活性与抗性的关系,为辅助家蚕育种及品种推广奠定基础.以DNS法测定不同品种海藻糖酶活性,以Fo-lion-酚法测定不同品种蛋白酶活性.以及以铜皂法测定脂肪酶活性,同时对不同品种家蚕4龄起蚕经口添加BmN-PV病毒进行抗性调查分析.结果表明,蛋白酶、脂肪酶酶活顺序为664×663较低,667×668相对较高,其他品种处于中间层次,对应抗性测定结果的相关分析表明,活性越强抗性越强,呈显著正相关关系;而海藻糖酶的活性与抗性的关系没有较好的相关性.因此,家蚕中肠消化液的蛋白酶、脂肪酶可能与家蚕对BmNPV病毒抗性有关.     

饲料蛋白质水平对克氏原螯虾幼体消化酶活性和肌肉成分的影响

用4种等能不同质量分数的蛋白质饲料(30%、35%、40%和45%)饲喂克氏原螯虾(Procambarus clarkii Girard)幼虾(5.46 g±0.51 g )28 d后,分析不同蛋白水平的饲料对克氏原螯虾幼虾肠道和肝胰脏消化酶活性和体成分的影响.结果表明:(1)饲料蛋白质浓度对脂肪酶和蛋白酶活性均有显著性影响(P<0.05);并随着饲料蛋白水平的提高,两酶活性都呈现先升高后降低的趋势.其中肠道蛋白酶和肝胰脏蛋白酶活性分别在饲料蛋白质浓度为40%和35%时最高;肠道和肝胰脏脂肪酶活性均在饲料蛋白质浓度为40%时最高,且各饲料组肝胰脏脂肪酶活性差异显著(P<0.05),而35%组和40%组之间的肠道脂肪酶活性无显著性差异(P>0.05).克氏原螯虾幼体的肠道和肝胰脏淀粉酶活性随饲料蛋白质水平的升高均呈波浪式变化;35%组与45%组之间的肠道淀粉酶活性和30%与45%组之间的肝胰脏淀粉酶活性均无显著性差异(P>0.05).(2)随着饲料中蛋白水平的升高,克氏原螯虾肌肉中蛋白质含量只有45%组显著升高(P<0.05),其他饲料组肌肉蛋白含量虽有上升趋势,但差异不显著(P>0.05).随着饲料蛋白浓度的增加,肌肉中脂肪含量反而下降,除30%组外,各组间差异显著(P<0.05).对克氏原螯虾肌肉灰分的测定表明,饲料蛋白浓度对肌肉中灰分含量的影响不显著(P>0.05).     

米黑根毛霉脂肪酶基因密码子优化及重叠延伸PCR合成

[目的]对米黑根毛霉脂肪酶基因的密码子进行优化,并采用重叠延伸PCR合成设计的基因序列。[方法]根据毕赤酵母(Pichiapastoris)密码子使用偏好性,对米黑根毛霉脂肪酶成熟肽基因进行全面密码子优化,根据优化后的氨基酸编码序列设计合成30条长度约为48 bp的寡核苷酸片段,并采用重叠延伸PCR法扩增合成全长基因序列。[结果]经凝胶电泳和酶切鉴定、测序分析表明,合成的目的基因与设计的序列相一致。[结论]该研究为脂肪酶基因在毕赤酵母中的构建及后续表达奠定了基础。     

Fat feeding increases equine heparin-released lipoprotein lipase activity

The aim of this study was to establish the dose-response relationship between fat intake and heparin-released plasma lipoprotein lipase (LPL) activity in horses. Eight mature trotters were fed 4 rations with different fat levels (3.0, 5.0, 7.7, or 10.8% fat in the dry matter) according to a 4 X 4 Latin square design. The experimental rations consisted of hay and different concentrates; the concentrates and hay were given in a 3:1 ratio on an energy basis. Soybean oil was added to the concentrates at the expense of isoenergetic amounts of glucose. Blood samples were taken at the end of each dietary period, which lasted 3 weeks. Fat feeding was found to increase heparin-released plasma LPL activity in a dose-dependent fashion. When the data from this study and previous studies were combined it was calculated that an increase in fat intake by 1 g/kg dry matter is associated with an increase in LPL activity by 0.98 micromol fatty acid released-mL(-1) x h(-1). Fat feeding raised the plasma concentrations of total cholesterol, high-density lipoprotein cholesterol, and phospholipids. Diet did not have a statistically significant effect on plasma triacylglycerol concentrations. The results are discussed in the light of the possible enhancing effect of fat feeding on the oxidative capacity of skeletal muscle.     

基因组步移技术扩增嗜水气单胞菌J-1株脂酶基因全长及原核表达

根据已发表的引物序列合成一对引物,以嗜水气单胞菌J-1株基因组为模板扩增脂酶基因,得到一个保守的383 bp基因片段。进一步应用基因组步移技术扩增其两侧未知序列,分别以DraⅠ,EcoRⅤ,PvuⅡ,ScaⅠ,StuⅠ,SmaⅠ6个内切酶酶切的基因组构建6个不同的基因组步移文库,再以基因组步移文库为模板,设计引物向已获得的保守片段两侧进行PCR扩增,将扩增到的两侧序列与保守片段拼接后得到一个3 476 bp的片段,通过DNA Star软件分析,找到一个2 415 bp的开放阅读框,经Blast软件分析,其与嗜水气单胞菌ATCC 7966脂酶、嗜水气单胞菌AH-3磷脂酶A1、嗜水气单胞菌H3脂酶、嗜水气单胞菌Mcc-2脂酶、嗜水气单胞菌J MP636磷脂酶C的序列同源性分别为97%、97%、88%、83%和82%。将DNA序列翻译成氨基酸序列后,发现其包含一个多肽序列(VHFLGHSLGA),这个序列在脂酶中高度保守。     

添加外源脂肪酶对瓦氏黄颡鱼的生长、消化酶及血清生化指标的影响

为考察添加脂肪酶酶制剂对瓦氏黄颡鱼生长、消化酶以及血清生化指标的影响,在基础饲料中分别添加0mg/kg、100mg/kg、300mg/kg、500mg/kg脂肪酶制剂,饲喂平均体重(3.47±0.05)g的瓦氏黄颡鱼90d。试验结果表明:添加脂肪酶100mg/kg、300mg/kg、500mg/kg组分别比对照组增重率提高3.86%(P>0.05)、9.29%(P<0.05)、6.75%(P>0.05),饲料系数降低了3.17%(P>0.05)、6.35%(P<0.05)、5.29%(P<0.05)。同时添加脂肪酶各组的血清甘油三酯、低密度脂蛋白和总胆固醇含量都显著低于对照组(P<0.05),通过消化酶测定表明,脂肪酶添加各组均显著提高了胰蛋白酶和肠淀粉酶活性(P<0.05),100mg/kg脂肪酶组还显著提高了胰脂肪酶和胰淀粉酶(P<0.05),300mg/kg脂肪酶组胰脂肪酶活性最高(P<0.05)。上述研究表明,脂肪酶在瓦氏黄颡鱼饲料中的适当添加量为300mg/kg为宜。     

Ontogenic development of digestive enzymes in bigfin reef squid (Sepioteuthis lessoniana)

Bigfin reef squid (Sepioteuthis lessoniana) is an economically important exported aquatic animal in Thailand, but little is known about its husbandry, which needs to be clarified. In this study, the ontogenic development of the main digestive enzymes (pepsin, trypsin, chymotrypsin, lipase and amylase) was investigated within 30 days after hatching (DAH). Fertilized eggs of the wild cuttlefish were hatched and then distributed into four replicate aquaria assigned to varying collection times (0, 5, 10, 15, 20, 25 and 30 DAH). The development of pepsin suggests acidic digestion functionality at 5 DAH, and its specific activity was maintained over the studied period. Specific activities of the serine proteases, trypsin and chymotrypsin, fluctuated significantly with similar pattern, and appear to be the key enzymes contributing ontogenic changes in protein catabolism. The specific activity of lipase was relatively high at 0 DAH, dramatically increased by 5 DAH, and then appeared constant until the end of observations, suggesting important role in utilizing the lipid from yolk at hatching, as well as probably in sparing protein utilization. Dramatic increase in amylase specific activity occurred by 10 DAH after which the activity was constant until 30 DAH, suggesting the utilization of carbohydrate as energy evolves comparatively late. Findings from the current study could be used to manage nutritional protocols according to the digestive enzyme patterns, as well as to develop artificial diets for rearing S. lessoniana.     

固定化微生物脂肪酶粉转化生物柴油研究

采用两种转酯方案进行脂肪酶粉转化生物柴油研究,游离脂肪酶在转酯方案一中转酯效率较高为90.15％,在转酯方案二中转酯效率为52.51％.通过壳聚糖和硅藻土对脂肪酶粉固定化并在转酯方案一中的研究发现,硅藻土固定化酶的平均转酯效率高于壳聚糖固定化酶.两种固定化脂肪酶的循环转酯试验表明,随循环次数的增加,水解酶活和转酯效率均逐渐降低,并呈现显著的相关性.