【作者】 于欣君；

【导师】 池振明；

【作者基本信息】 中国海洋大学 ， 微生物学， 2011， 博士

【摘要】 酸性蛋白酶,又叫天冬氨酸蛋白酶,是一种在酸性环境下可以将蛋白质分解成多肽或氨基酸的酶类。近年来,酸性蛋白酶广泛应用于生产,特别在乳制品、饲料工业和酒精发酵等方面,具有很大的规模。近年来,由于牛凝乳酶供应量低,价格高和安全性等问题,来自其它物种,如细菌和真菌的牛奶凝固酶作为牛凝乳酶的替代品广泛引起人们的注意。解脂耶罗维亚酵母可以自然分泌大量的蛋白,具有多种用于遗传标记和分子工具,该酵母被认为是安全的。所以能利用解脂耶罗维亚酵母菌表达生产酸性蛋白酶取代牛凝乳酶具有重要的实际意义。本研究发现扣囊复膜酵母菌（Saccharomycopsis fibuligera）A11菌株具有酸性蛋白酶的活性,但该酶是胞壁酶,不能分泌到胞外。所以为了研究该酶的性质和该酶在牛奶凝固中的应用,从扣囊复膜酵母菌（Saccharomycopsis fibuligera）A11菌株中克隆编码该胞壁酸性蛋白酶的基因APG,并且在解脂耶罗维亚酵母（Yarrowia lipolytica）Po1h菌株中表达,获得了高重组酸性蛋白酶活性的菌株71。纯化了胞外的重组酸性蛋白酶,并研究其酶学性质。发现该酶分子量大约99 kDa,其最适pH和温度分别是3.5和33℃,并且该酶在pH 1.0到3.0的范围内稳定。该重组酶活性可被Zn²⁺激活,但被Hg⁺、Fe²⁺、Fe³⁺、Mg²⁺、EDTA、碘乙酸和胃酶抑素等抑制。来自重组子71的重组酸性蛋白酶具有很高的牛奶凝固活性,说明可将其作为牛凝乳酶的替代物应用于奶酪工业。同时把该酸性蛋白酶基因连接到表面展示质粒pINA1317-YlCWP110的多克隆位点中,通过该质粒将酸性蛋白酶展示在解脂耶罗维亚酵母Po1h的细胞表面上,其中展示蛋白酶的细胞在牛奶平板上可以形成透明圈,说明它们具有胞外酸性蛋白酶活性,并且展示酸性蛋白酶的细胞可用于牛奶凝固过程中。并筛选得到无His标签、带6His标签和8His标签的转化子20、20和6,酶活分别是63.7U/OD_600nm、42.0 U/OD_600nm和25.3U/OD_600nm。这些结果说明无His标签的酸性蛋白酶酶活性较高,在实际应用中展示在酵母表面上的酸性蛋白酶不用His标签。将上述转化子应用于牛奶凝固试验中,并优化了牛奶凝固的条件。在pH 3.0, 40°C,加入20 mM的CaCl₂和10%（w/v）脱脂奶粉的条件下,牛奶凝固活性达到最大的1142.9 U/mL。说明酵母细胞表面展示的酸性蛋白酶可以有效地应用于牛奶凝固。相对于分泌的酸性蛋白酶,表面展示的酸性蛋白酶省去了酶的纯化等过程,可以直接将表面展示有酸性蛋白酶的酵母细胞应用于牛奶凝固过程中,节约了生产时间和成本。所以,表面展示的酸性蛋白酶在食品和奶酪工业中具有很大的潜在应用价值。本研究是关于展示酸性蛋白酶的酵母细胞作用于牛奶凝固的首次报道。目前,发现不同来源的酵母菌具有很高的蛋白含量,可以用于单细胞蛋白的生产。单细胞蛋白可用于动物的饲料以及人类的食物。源于海洋鱼类肠道的解脂耶罗维亚酵母22a-2,每100g干细胞中含有47.6g蛋白,所以具有用于单细胞蛋白的潜在价值。为了使解脂耶罗维亚酵母22a-2既可以生产单细胞蛋白,又可以生产重组酸性蛋白酶用于牛奶凝固,将来自扣囊复膜酵母菌A11菌株的酸性蛋白酶基因在解脂耶罗维亚酵母22a-2中表达,所产的重组蛋白酶用于牛奶凝固中,并且重组酵母菌仍保持很高的蛋白含量。由该重组酸性蛋白酶水解酪蛋白产生的蛋白质水解产物与牛凝乳酶水解酪蛋白产生的蛋白质水解产物在大小方面一致,说明该重组酸性蛋白酶凝固牛奶的机理可能与牛凝乳酶的一致。更多还原

【Abstract】 The acid protease, also known as aspartic protease, is a sort of enzyme which can break down protein into polypeptide or amino acid. Recently, the acid protease is widely applied in many fields of industry,such as dairy, feed and fermentation.Milk coagulation is a basic step in cheese manufacturing. The calf rennet, the conventional milk-clotting enzyme obtained from the fourth stomach of suckling calves, has been the most widely used as the coagulant in cheese making to manufacture most cheese varieties. However, milk-clotting enzymes from alternative sources, such as bacteria and fungi, are used as the calf rennet substitutes and have received increasing attention due to the consumer constraints on the use of rennet, reduced supply and the increased price of calf rennet, in addition to the ever-increasing cheese production and consumption. It has been well documented that Yarrowia lipolytica appears as one of the most attractive microorganisms because it has naturally secreting high amount of proteins and a large range of genetic markers and molecular tools. The yeast has been used in several industrial processes and is non-pathogenic （GRAS, generally regarded as safe）. The Y. lipolytica expression/secretion system has been validated by the efficient production of active proteins of various origins.It has been found that Saccharomycopsis fibuligera A11 strain can produce acid protease. As this acid protease is a cell-bound enzyme and can’t be secreted into media, it is very difficult to purify and characterize it. So the gene encoding an acid protease natively produced by Saccharomycopsis fibuligera was cloned and overexpressed in Yarrowia lipolytica and the resultant recombinant acid protease was purified and characterized. The molecular mass of the purified enzyme was estimated as 99 kDa by gel filtration chromatography. The optimal pH and temperature of the purified acid protease were 3.5 and 33℃, respectively, and the enzyme was very stable over a pH range of 1.0-3.0. The recombinant acid protease was activated by Zn²⁺, but was inhibited by Hg⁺, Fe²⁺, Fe³⁺, and Mg²⁺, EDTA, EGTA, iodoacetic acid, and pepstatin. The purified recombinant acid protease from the positive transformant 71 had high milk clotting activity, suggesting that it may be used as a rennet substitute in the cheese industry.While the gene was cloned into the multiple cloning site of the surface display vector pINA1317-YlCWP110 and expressed in the cells of Yarrowia lipolytica, the cells displaying the acid protease could form clear zone on the plate-containing milk indicating that they had extracellular acid protease activity. The cells displaying the acid protease can be used to effectively clot skimmed milk. The highest clotting milk activity （1,142.9 U/ml） was observed under the conditions of pH 3.0, 40°C, 20 mM of CaCl₂, and 10% skimmed milk powder. We found that the acid protease displayed on the cells of Y. lipolytica which has generally regarded as safe status could be easily isolated and concentrated compared to the free acid protease. Therefore, the displayed acid protease may have many potential applications in food and cheese industries. This is the first report that the yeast cells displaying the acid protease were used to clot milk.In recent years, many yeast strains from different sources have been found to contain high content of protein and can be used as single protein. The single cell protein can be utilized as feed for animals and food for human. In order to use Y. lipolytica 22a-2 as both the potential single cell protein and producer of the recombinant acid protease for milk clotting, the acid protease gene from S. fibuligera A11 was expressed in the cells of Y. lipolytica 22a-2 and the produced acid protease was used to clot the skimmed milk. It was also found that Y. lipolytica 22a-2 producing acid protease still contained high content of protein. We observed that hydrolysis products fromκ-casein under catalysis of the recombinant acid protease were similar to those fromκ-casein under catalysis of the calf rennet. This may suggest that mechanisms of action of the recombinant acid protease are similar to those of action of the calf rennet.更多还原