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。     

脂肪酶及其在饲料工业中的应用研究

脂肪酶是一种重要的工业用酶,广泛应用于食品、纺织、化工、医药、饲料和能源等领域。目前,脂肪酶应用于饲料工业的研究相对较少。文中就脂肪酶近年来在饲料工业中的应用研究做简要的综述,为今后脂肪酶在饲料中的开发应用提供参考。     

Effect of water temperature on the growth performance and digestive enzyme activities of Chinese longsnout catfish (Leiocassis longirostris Günther)

The present study was carried out to investigate the influence of water temperature on the growth performance and digestive enzyme (pepsin, trypsin and lipase) activities of Chinese longsnout catfish. Triplicate groups of Chinese longsnout catfish (35.6±0.48 g, mean±SE) were reared at different water temperatures (20, 24, 28 and 32 °C). The feeding rate (FR), specific growth rate (SGR) and feed efficiency ratio (FER) were significantly affected by water temperatures and regression relationships between water temperature and FI, SGR as well as FER were expressed as FR=−0.016 T ²+0.91 T −10.88 ( n =12, R ²=0.8752), SGR=−0.026 T ²+1.39 T −17.29 ( n =12, R ²=0.7599) and FER=−0.013 T ²+0.70 T −8.43 ( n =12, R ²=0.7272). Based on these, the optimum temperatures for FR, SGR and FER were 27.66, 26.69 and 26.44 °C respectively. The specific activities of digestive enzymes at 24 or 28 °C were significantly higher than that at 20 or 32 °C. In addition, there was a significant linear regression between FR or SGR and specific activities of pepsin and lipase, which indicated that pepsin and lipase played important roles in regulating growth through nutrient digestion in Chinese longsnout catfish.     

Development of Digestive Enzymes in California Halibut <Emphasis Type="Italic">Paralichthys californicus</Emphasis> Larvae

California halibut Paralichthys californicus is an important commercial species with high aquaculture potential in Baja California Sur, México. To optimize the feeding process using live prey and/or inert diets, we evaluated alkaline proteases, pepsin, trypsin, chymotrypsin, leucine aminopeptidase, lipase, α-amylase, and acid and alkaline phosphatase activities on starved larvae and larvae fed live prey. Highest activities were observed for alkaline protease, trypsin, chymotrypsin, leucine aminopeptidase, and alkaline phosphatase in feeding larvae than starved larvae on day 4 after hatching. At day 5, a sizeable increase in all enzymatic activities was detected in feeding larvae. Alkaline protease, trypsin, chymotrypsin, and alkaline phosphatase decreases progressively from day 5 until day 18. At day 18, a slight pepsin activity was observed. This was considered an indicator of the start of digestive system maturation. We concluded that total enzymatic equipment for this species is complete between day 18 and 30 after hatching. Based on this evidence, early weaning from live prey to inert feed would be possible at this time.     

Serum lipase determination in the dog: a comparison of a titrimetric method with an automated kinetic method

An enzymatic, kinetic method for determining serum lipase activity was evaluated and compared to a standard manual method for use in dogs. The kinetic method was a commercial kit adapted for use on a tandem access clinical chemistry analyzer and utilized a series of coupled enzymatic reactions based on the hydrolysis of 1,2-diglyceride by lipase. The manual method was the Cherry-Crandall technique based on the titration of base against the acid formed by hydrolysis of an olive oil substrate by lipase. The correlation between the two methods was very good (r = 0.94). The reference range for 56 clinically healthy dogs assayed by the kinetic method was 90 to 527 U/L. Diseases associated with a greater than twofold elevation in serum lipase activity as determined by the kinetic method included pancreatitis, gastritis with liver disease, and oliguric renal failure with metabolic acidosis. In some cases, pancreatitis was seen with other clinical problems, such as gastroenteritis, diabetic ketoacidosis, duodenal mass, disseminated intravascular coagulation, and septic peritonitis. Diseases associated with serum lipase activity within the reference range or elevated less than twofold included gastritis, gastric ulcer, cholestasis, phenobarbital-induced hepatopathy, colitis, copper hepatopathy, abdominal hematoma, apocrine gland adenocarcinoma, and thrombocytopenia with pneumonia.     

Anti-diabetic effects of emodin involved in the activation of PPARγ on high-fat diet-fed and low dose of streptozotocin-induced diabetic mice

Rheum palmatum Linn has been widely applied in the clinical treatment of diabetes mellitus. It has been found that emodin as the major bioactive component of R. palmatum L exhibits the competency to activate peroxisomal proliferator-activated receptor-γ (PPARγ) in vitro. So the aim of this study was to evaluate the anti-diabetic effects of emodin through the activation of PPARγ on high-fat diet-fed and low dose of streptozotocin (STZ)-induced diabetic mice. The diabetic mice were intraperitoneally injected with emodin for three weeks. No changes of food consumption and the body weight in emodin-treated mice were monitored daily during the entire experiment. At the end of experiment, the levels of blood glucose, triglyceride and total cholesterol in serum were significantly decreased after emodin treatment. However, serum high-density lipoprotein cholesterol (HDLc) concentration was significantly elevated. The glucose tolerance and insulin sensitivity in emodin-treated group were significantly improved. Furthermore, the results of quantitative RT-PCR analysis showed that emodin significantly elevated the mRNA expression level of PPARγ and regulated the mRNA expressions of LPL, FAT/CD36, resistin and FABPs (ap2) in liver and adipocyte tissues. No effects on the mRNA expressions of PPARα and PPARα-target genes were observed. Taken together, the results suggested that the activation of PPARγ and the modulation of metabolism-related genes were likely involved in the anti-diabetic effects of emodin.     

米黑根毛霉脂肪酶在毕赤酵母中的高效表达及酶学性质研究

根据毕赤酵母(Pichia pastoris)密码子使用偏爱性,对米黑根毛霉脂肪酶(RML)成熟肽基因进行全面密码子优化,以提高RML基因在酵母细胞中表达的水平,并对重组脂肪酶的酶学性质进行了研究。运用重叠延伸PCR合成优化后的RML成熟肽基因,并进一步构建毕赤酵母表达载体p PIC9K-RML,通过电击法转化毕赤酵母GS115菌株,经G418抗性和PCR筛选获得高拷贝转化重组子。重组酵母菌用0.5%甲醇诱导异源基因表达,在28℃摇瓶培养168 h后酶活力达到122 U/m L,较未优化的原始菌株的表达酶活提高了10倍。重组脂肪酶最适温度为45℃,最适p H值为7,甲醇体积分数为40%以下时重组RML具有较好的稳定性。     

固定化酶载体的研究现状及其应用

研制开发性能优异的固定化载体,已成为固定化酶技术最为活跃的研究方向之一。固定化酶在生物及生物工程、环境化工、食品、医药等领域得以迅速发展,其中,结合现代分析仪器,固定化酶反应器(IMER)将成为固定化酶应用的一个新领域。本文对近几年来固定化酶载体的制备及固定化酶的应用进行了综述。