产脂肪酶菌株的筛选、鉴定以及酶学性质的研究

【目的】筛选高产脂肪酶菌株并鉴定其种属,研究脂肪酶的酶学性质.【方法】以橄榄油为唯一碳源对富含油污土壤中产脂肪酶微生物进行富集培养,用中性红平板进行初筛,结合改进铜皂分光光度计法测定酶活力;对筛选的高产脂肪酶菌株进行形态学观察和相关生理生化实验,再结合16S rDNA序列分析确定其种属;确定该菌株所产脂肪酶的最适作用温度和pH值,并研究金属离子、有机溶剂以及表面活性剂对酶活的影响.【结果】筛选得到一株高产脂肪酶菌株LZ-5,其所产脂肪酶的酶活力为4.78 U/mL;菌种鉴定为表皮葡萄球菌(Staphylococcus epidermidis).该酶最适作用温度为40℃,最适pH值为9.0;Ca~(2+)、Mg~(2+)对酶活力有促进作用,Fe~(2+)、Zn~(2+)、EDTA、SDS、甲醇和乙醇对酶活力有抑制作用,对Na~+、K~+、丙三醇的耐受力较高.【结论】成功分离一株表皮葡萄球菌高产脂肪酶菌株.     

Breed associations for canine exocrine pancreatic insufficiency

BACKGROUND: Knowledge of breed associations is valuable to clinicians and researchers investigating diseases with a genetic basis. HYPOTHESIS: Among symptomatic dogs tested for exocrine pancreatic insufficiency (EPI) by canine trypsin-like immunoreactivity (cTLI) assay, EPI is common in certain breeds and rare in others. Some breeds may be overrepresented or underrepresented in the population of dogs with EPI. Pathogenesis of EPI may be different among breeds. ANIMALS: Client-owned dogs with clinical signs, tested for EPI by radioimmunoassay of serum cTLI, were used. METHODS: In this retrospective study, results of 13,069 cTLI assays were reviewed. RESULTS: An association with EPI was found in Chows, Cavalier King Charles Spaniels (CKCS), Rough-Coated Collies (RCC), and German Shepherd Dogs (GSD) (all P < .001). Chows (median, 16 months) were younger at diagnosis than CKCS (median, 72 months, P < .001), but not significantly different from GSD (median, 36 months, P = .10) or RCC (median, 36 months, P = .16). GSD (P < .001) and RCC (P = .015) were younger at diagnosis than CKCS. Boxers (P < .001), Golden Retrievers (P < .001), Labrador Retrievers (P < .001), Rottweilers (P = .022), and Weimaraners (P = .002) were underrepresented in the population with EPI. CONCLUSIONS AND CLINICAL IMPLICATIONS: An association with EPI in Chows has not previously been reported. In breeds with early-onset EPI, immune-mediated mechanisms are possible or the disease may be congenital. When EPI manifests later, as in CKCS, pathogenesis is likely different (eg, secondary to chronic pancreatitis). Underrepresentation of certain breeds among dogs with EPI has not previously been recognized and may imply the existence of breed-specific mechanisms that protect pancreatic tissue from injury.     

长薄鳅幼鱼消化道指数和消化酶活性研究

测定了长薄鳅(Leptobotia elongate Bleeker)幼鱼(8.0～110.1 g)的消化道指数和不同体重组(10 g、30 g、50 g)幼鱼的胃、肝胰脏、前肠、中肠、后肠的淀粉酶、胃蛋白酶、胰蛋白酶及脂肪酶活性。结果:(1)长薄鳅幼鱼肝指数平均为(1.400±0.004)%,比肠长平均为(0.418±0.080),体重与体长呈幂函数相关,相关式为:W=0.019L2.818 1(R2=0.948,P<0.01,n=43);鱼体肥满度在30 g组中最高。(2)各部位消化酶活性均以蛋白酶>脂肪酶>淀粉酶;各体重组淀粉酶活性在肝胰脏中最高(P<0.05);蛋白酶活性在胃和中肠中较高;脂肪酶活性沿消化道从前至后升高;(3)各部位淀粉酶活性在各体重组间变化不大,胃蛋白酶、肠段胰蛋白酶和脂肪酶活性随体重增加而下降。本研究认为长薄鳅幼鱼为杂食、偏肉食性,其消化酶活性存在组织特异性,体重30 g左右是其营养需求转变或者生长发育的重要时期。     

Effect of Jia-jian-yi-yin decoction on endothelial dysfunction in ovariectomized female rats

AIM: To investigate the effect of Jia-jian-yi-yin decoction on endothelium-dysfunction in ovariectomized rats. METHODS: The ovariectomized rats were treated with Jia-jian-yi-yin decoction or turbid liquid of estradiol valerate for 8 weeks. The vascular ring tension was measured. Scanning electron microscopy and Western blotting were applied to assess the function of endothelium-dependent dilation, aortic endothelial morphology and the expression of endothelial lipase. The pathologic sections were prepared to observe the effect of Jia-jian-yi-yin decoction on the uterus. RESULTS: In ovariectomized rats, the decrease in endothelium-dependent relaxation to acetylcholine (ACh) was reversed to normal level, the endothelial morphology returned to normal without lipid accumulation and the endothelial lipase expression was decreased by Jia-jian-yi-yin decoction. Furthermore, no obvious change of the wet weight of uterine between the ovariectomized rats with or without Jia-jian-yi-yin decoction treatment was observed. CONCLUSION: Jia-jian-yi-yin decoction may have protective effects on endothelium-dependent vasodilation and aortic endothelial morphology in estrogen-deficient animals.     

低温脂肪酶产生菌的筛选及发酵条件研究

通过使用RodamineB平板筛选法,从新疆具有典型低温环境地区的近200份土样中筛选分离到11株产低温脂肪酶的菌株。其中1株酶活较高的菌株L22,根据形态观察初步断定为假丝酵母,该酶的最适反应温度为30℃,属低温脂肪酶。摇瓶发酵确定该菌株产脂肪酶的最适发酵培养条件为:葡萄糖0.5%,豆饼粉2%,玉米浆2%,KB2BHPOB4B0.5%,NaNOB3B0.5%,橄榄油0.25%,初始pH值为7,发酵温度为20℃,摇床培养48h,酶活最高可达7.17U/mL,添加适量的油脂对产脂肪酶有诱导作用。     

酶法提取芦笋皮中高活性膳食纤维的研究

以芦笋皮为原料，采用酶法水解制备高活性膳食纤维。试验结果表明：用0.54%的脂肪酶(100 U/mg)、0.80%的淀粉酶(25 U/mg)和0.80%的糖化酶(20 U/mg)、1.20%的蛋白酶(100 U/mg)可提取出高活性的膳食纤维。所得膳食纤维纯度高，总膳食纤维含量达83.08%，其中水溶性膳食纤维17.02%，不溶性膳食纤维66.06%。而且生理活性好，持水性和溶胀性分别为886%和4.5 mL/g。     

肉碱对中华鳖脂肪代谢的影响

选用1 170只211日龄中华鳖进行养殖试验,研究不同剂量(0,50,100 mg/kg)的l-肉碱对中华鳖脂肪代谢的影响. 通过解剖取样测定肝脏、四肢肌肉和脂肪块的脂肪代谢酶活性及肌细胞内脂肪酸β-氧化反应等生化指标,探讨其作用机理. 结果表明,在饲料中添加100 mg/kg l-肉碱显著降低了中华鳖四肢的脂肪沉积量的27.11%(P<0.01),肝脏和脂肪中的苹果酸脱氢酶及脂肪中激素敏感脂酶总活力分别提高了24.32%(P<0.01)、32.22%(P<0.01)和14.88%(P<0.05),肌肉和脂肪中脂蛋白脂酶总活力分别提高了27.08%(P<0.01)和13.18%(P<0.05),肌肉中游离肉碱、总酸不溶肉碱含量分别升高了16.92%(P<0.01)和38.24%(P<0.01).     

Inhibitory effect of 5-fluorouracil on ultra-microstructure and insulin secretion of pancreatic β cells

AIM: To investigate the molecular mechanisms of β cell dysfunction induced by 5-fluorouracil (5-FU) in islet β cell line (NIT-1 cells). METHODS: The NIT-1 cells were treated with different concentrations of 5-FU. The content of insulin in the culture medium was determined by radioimmunoassay. Cell apoptosis was observed by flow cytometry with annexin V/PI staining. The ultra-microstructural changes of NIT-1 cells were observed under transmission electron microscope. The expression of pancreatic and duodenal homeobox protein 1(PDX-1) at mRNA and protein levels in NIT-1 cells was examined by RT-PCR and Western blotting, respectively. RESULTS: Exposed to the low glucose concentration (5.6 mmol/L), insulin secretion in NIT-1 cells was not significantly decreased following a 24 h treatment with 5.0 to 40.0 mg/L 5-FU (P>0.05). On the contrary, the high glucose (16.7 mmol/L)-stimulated insulin secretion in NIT-1 cells was inhibited by 5.0 to 40.0 mg/L of 5-FU in a dose-dependent manner after 24 h of incubation (P<0.01). The apoptosis rate of NIT-1 cells was significantly increased as compared to those in the control levels(P<0.05). The structural changes of mitochondria were the main apoptotic changes under transmission electron microscope. Significant down-regulation of PDX-1 expression at mRNA and protein levels was observed in NIT-1 cells treated with 5-FU at the concentration of 10.0 mg/L to 40.0 mg/L(P<0.05).CONCLUSION: 5-FU inhibits the insulin secretion in islet β cell induced by high glucose. A relative deficiency in insulin secretion following 5-FU treatment is related to the changes of β cell ultra-microstructure and the reduction of β cell numbers, by which an increase in apoptosis of pancreatic β cells is induced. Down-regulation of PDX-1 expression may play a pivotal role in increasing the apoptosis of pancreatic β cells induced by 5-FU in high-glucose condition.     

南极假丝酵母脂肪酶B的修饰与改造

南极假丝酵母脂肪酶B是用途最为广泛的脂肪酶之一,可用于许多有机化合物、医药中间体等的合成。为了改善南极假丝酵母脂肪酶B的催化性能,提高其环境适应性,同时提高蛋白的表达量,降低生产成本,从而使其更适于工业化应用,许多针对酶蛋白分子的修饰及改造方法已在南极假丝酵母脂肪酶B中得到大量应用。本文从化学修饰、理性设计和非理性设计3个方面详细综述了南极假丝酵母脂肪酶B的修饰与改造的关键技术,包括定点突变、蛋白质融合、易错PCR和DNA改组等,同时也介绍了以上技术在改良南极假丝酵母脂肪酶B特性方面的诸多成功实例。     

低温热处理对超高温灭菌乳中假单胞菌脂肪酶的活性影响

通过分析不同因素对假单胞菌脂肪酶活性的影响以及假单胞菌脂肪酶活性与超高温灭菌(UHT)乳脂肪上浮的关系,研究了控制UHT乳中假单胞菌脂肪酶活性和延缓脂肪上浮现象的措施。结果表明pH值对假单胞菌脂肪酶的活性有显著性影响(P<0.01),50、55、60℃(20min)的低温热处理能显著降低假单胞菌脂肪酶的活性(P<0.01)。贮藏试验表明低温热处理对控制UHT乳在贮存过程中的假单胞菌脂肪酶活性水平(P<0.01)和脂肪上浮现象(P<0.05)有明显的作用,而55℃和15min的低温热处理对缓解UHT乳在贮藏过程中的脂肪上浮现象最为有效。