利用生物技术开发一种新乳糖酶及其高效生产途径
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摘要
意义和目的
牛乳及乳制品含有丰富的优质蛋白质、脂肪、碳水化合物以及几乎全部已知的维生素和多种矿物质，尤其是其钙含量高且钙磷比例适当，易被人体消化吸收，还含有免疫球蛋白等抗病因子，因此牛乳及乳制品是人类改善营养、增强体质不可缺少的理想食品。我国已于2000年11月15日正式启动“学生饮用奶计划”，旨在提高全民营养状况、推动我国农业经济发展。目前世界人均牛乳年消费量为94kg，亚洲人均40kg，而我国仅为7kg，相差甚远，除了经济发展水平以外，一个重要的原因就是在中国人群中有很大一部分患有乳糖不耐受症。乳糖是牛乳中主要糖成分，其含量约为5％左右。许多人(因人种而异，西欧占2-8％，亚洲、非洲占60-90％)体内缺乏乳糖酶，因此他们很难消化牛奶，饮用牛奶后，乳糖在肠道内，被肠道细菌分解发酵，产生大量二氧化碳气体，导致肠道膨胀，并兴奋肠道蠕动，使收缩加强，造成肠鸣和腹泻，这种疾病称为乳糖不耐受症。生产低乳糖乳及乳制品是解决乳糖不耐受症问题的最佳途径。
β-半乳糖苷酶(β-D半乳糖苷半乳糖水解酶，β-D-galactoside galatohydrolase,EC 3.2.1.23)通常被称为乳糖酶(Lactase)。该酶能将乳糖水解为半乳糖和葡萄糖，并具有半乳糖苷的转移作用。乳糖酶广泛存在于植物、细菌、真菌、放线菌以及动物肠道中。乳糖酶主要用来治疗乳糖不耐受症，处理加工牛乳、乳清，生产低乳糖牛奶和乳制品。近几年随着人们对乳糖不耐受症认识的深入，乳糖酶在食品和乳制品工业中的作用日益显著。但目前乳糖酶并未在中国食品和乳制品工业中得到广泛应用，主要原因是：(1)乳糖酶的单位产量较低；(2)市场上销售的乳糖酶多为胞内酶，酶的耐热性差、适用范围窄；且这种酵母来源(Kluyveromyces lactis、Kluyveromyces fragilis)的胞内酶，需破碎细胞才能得到，提取、纯化工艺繁杂。由于上述等原因致使乳糖酶销售价格过高、应用成本昂贵、适用范围窄，不能满足食品和乳制品工业多方面的需要。
本论文研究的目的是从本实验室保存的一株产乳糖酶的亮白曲霉(Aspetgillus candidus)中克隆并获得拥有自主知识产权的乳糖酶基因，构建高效表达、有效分泌乳糖酶的重组毕赤酵母生物反应器，以解决乳糖酶单位产量低、胞内酶提取困难的问题。期望利用生物技术开发一条乳糖酶高效生产的新途径。
中国农业科学院博士学住论文
结论
1．本研究从一株产乳糖酶的亮白曲霉（ASpergillu candidus冲克隆得到了乳糖
酶基因组DNA及CDN＊序列，序列分析表明，该乳糖酶基因组DNA序列长3458hp，
其中含有8个内含子，CDNA编码区长3015hp，共编码1005个氨基酸，前19个氨
基酸为信号肽序列，氨基酸序列中共含有11个潜在的糖基化位点。将不同来源的
乳糖酶基因与此基因进行比较发现，该基因与绝大多数乳糖酶基因同源性较低；与
商业化应用的、在本研究中作为对照的 A．o叭ae ATCC 20423的乳糖酶基因组 DNA
和衍生的氨基酸序列比较，两基因CDNA的同源性为99％，氨基酸的同源性为
99．5％，二者的基因组DNA序列中有19个碱基不同，内含子区域有5个碱基不同，
CDNA序列中有 14个碱基不同，其中 3个碱基的变化引起了 3个氨基酸的改变：ZI＋，
T从cry a幻一？U．can＆血小 牡0，1U．口W二a一～＊U．can”山小989，NU．O炒二ae
—D（．candidusX但经实验证实，二者在酶学性质上有显著的差异，由此说明我们
克隆到了一个新基因，我们己将该基因在 GENEBANK中登录，登录号为：EMBL
ACCESSIONNO．AJ431643。
2．研究了亮白曲霉的乳糖酶的酶学性质：其最适叫．2，最适温度为60OC，PH
稳定范围在pH3习之间，比活为706．54U／mg，以ONPG为底物时的义为
1石7mmol几，七x为3．33卜mo厂L。将亮白曲霉的乳糖酶与商业化应用的、本研究作
为对照的米曲霉ATCC 20423的乳糖酶进行了酶学性质的比较，结果表明亮白曲霉
的乳糖酶在热稳定性、金属离子稳定性、比活、（、pH范围方面均优于后者，该乳
糖酶具有更优越的酶学性质，在工业化生产中具有更广阔的应用前景。
3．亮白曲霉的乳糖酶基因在大肠杆菌和毕赤酵母中均得到了表达，大肠杆菌
表达的乳糖酶蛋白分子量约为 11 okD，与理论分子量山 okD湘符；毕赤酵母表达的
乳糖酶蛋白分子量为120．130kD，二者分子量之间有差别的原因可能是毕赤酵母表
达系统可对该蛋白进行翻译后的糖基化修饰作用，而原核表达系统不具备该功能。
毕赤酵母表达系统可高效地表达有生物活性的亮白曲霉的乳糖酶，分泌表达量
近 6g／L，酶活为 3600U／InL，且目的蛋白占发酵液中总分泌蛋白的 90％以上，发酵
液中杂蛋白含量很少。因此本研究开辟了一条产量高、分离纯化简便、廉价大规模
生产乳糖酶的新途径。
4．初步进行了亮白曲霉的乳糖酶水解新鲜牛奶的应用效果研究。将不同酶量
的孪糖酶O000U、1500U）分另添力到10mL牛奶中，测定在不同温度下OZ℃、37
OC、50oC、60oCX不同时间内，乳糖酶对牛奶中的乳糖的水解程度。通过高压液?
The enzyme beta~D-galactosidase(beta-D-galactoside galactohydrolase, EC 3.2.1.23, also referred to as lactase)hydrolysis the disaccharide lactose to glucose and galactose. It is widely distributed various microorganisms such as bacteria, fungi, and also in various plants and animals intestine. Lactase can be used for treatment of "lactose intolerance", and production of low-lactose milk, whey and other dairy products. Although lactase has important applications in the fields of medicine, dairy industry, some problems obstructs large-scale industrial production of lactase. Main problems ate: (l)Low production and Enzymatic activity of lactase; (2)Commercial lactase mainly derived from yeasts(Kluyveromyces lactis <. Kluyveromyces fragilis) with low thermostability and narrow pH range; (3)An intracellular lactase resulting in the high cost due to extraction and purification processes.
The purposes of our research are: (1) Screening lactase-yielded strain with superior enzymatic characteristics, identifying and isolating the lactase gene from the microorganism. (2) Enhancing production of lactase and reducing production cost via constructing high effective bioreactor of Pichia pastoris which can express and secrete lactase effectively.
The results obtained as follows:
1. A lactase-yielded strain Aspergillus candidus is screened. Genomic DNA and cDNA sequences of lactase from Aspergillus candidus are cloned. Sequences analysis reveals that the genomic DNA with the length of 345 8bp containing eight introns, cDNA with the length of 3015bp encoding a polypeptide of 1005 amino acid residues. Signal peptide is consisted of 19 amino acid residues. 11 potential N-glycosylation sites are assumed. Comparing the gene cDNA and the deduced amino acid sequences with the lactase sequences from various organism sources shows a very low homology, and the highest homology of cDN A and the deduced amino acid sequence are observed between the lactase of Aspergillus candidus and from Aspergillus oryzae ATCC 20423 which used as control strain. Homology of cDNA sequence is 99%, and homology of the deduced amino acid sequence is 99.5%. Although only 3 amino acid residues are
different in the lactase from A. candidus2, 1(A. oryzae P (A. candidus); 420, l(A. ory~ae)^M(A. candidus); 989, N(^. oryzae-℃D(A. candidus)}, the characteristics of the enzyme exhibits obvious difference from that of A. oryzae. The lactase gene from Aspergillus candidus is a new gene for food and diary industry.
2. The lactase from Aspergillus candidus shows optimum pH of 5.2, optimum temperature of 60℃, and the stable pH ranging from 3.0 to 9.0. Specific activity is 706.54U/mg. The Km is 1.67mmol/L, Fraax is 3.33umol/L with ONPG as a substrate. The enzymatic characteristics of Aspergillus candidus is superior in many aspects to that of Aspergillus oryzae ATCC 20423 including the thermostability, specific activity, metal ion stability, Km, and pH stable range.
3. Lactase gene from Aspergillus candidus is expressed in E. coil and Pichia pastoris. By SDS-PAGE, the molecular weight of lactase expressed in E. coil is estimated to be about 11 OkD according with calculated molecular weight. The molecular weight of lactase protein expressed in Pichia pastoris is about 120-13 OkD probably due to glycosylation. Lactase from Aspergillus candidus is expressed in Pichia pastoris efficiently. High-level secreted lactase is about 6g/L with enzymatic activity of 3600U/mL. Therefore, the research open a new and convenient avenue for high-yielded lactase production.
4. 3000U and 1500U of lactase is added separately in lOmL milk and the lactose content in milk by HPLC at the condition of various temperature (12℃ > 37 ℃ > 50 ℃ .. 60 ℃) and various incubated times is determined. The result shows, (1) lactose hydrolysis rate under the condition of 3000U lactase is higher than that of 1500U at the same temperature and incubated tune. (2) The lactose hydrolysis rate is higher at the higher temperature than that of lower temperature at the same incubated time. The
牛乳及乳制品含有丰富的优质蛋白质、脂肪、碳水化合物以及几乎全部已知的维生素和多种矿物质，尤其是其钙含量高且钙磷比例适当，易被人体消化吸收，还含有免疫球蛋白等抗病因子，因此牛乳及乳制品是人类改善营养、增强体质不可缺少的理想食品。我国已于2000年11月15日正式启动“学生饮用奶计划”，旨在提高全民营养状况、推动我国农业经济发展。目前世界人均牛乳年消费量为94kg，亚洲人均40kg，而我国仅为7kg，相差甚远，除了经济发展水平以外，一个重要的原因就是在中国人群中有很大一部分患有乳糖不耐受症。乳糖是牛乳中主要糖成分，其含量约为5％左右。许多人(因人种而异，西欧占2-8％，亚洲、非洲占60-90％)体内缺乏乳糖酶，因此他们很难消化牛奶，饮用牛奶后，乳糖在肠道内，被肠道细菌分解发酵，产生大量二氧化碳气体，导致肠道膨胀，并兴奋肠道蠕动，使收缩加强，造成肠鸣和腹泻，这种疾病称为乳糖不耐受症。生产低乳糖乳及乳制品是解决乳糖不耐受症问题的最佳途径。
β-半乳糖苷酶(β-D半乳糖苷半乳糖水解酶，β-D-galactoside galatohydrolase,EC 3.2.1.23)通常被称为乳糖酶(Lactase)。该酶能将乳糖水解为半乳糖和葡萄糖，并具有半乳糖苷的转移作用。乳糖酶广泛存在于植物、细菌、真菌、放线菌以及动物肠道中。乳糖酶主要用来治疗乳糖不耐受症，处理加工牛乳、乳清，生产低乳糖牛奶和乳制品。近几年随着人们对乳糖不耐受症认识的深入，乳糖酶在食品和乳制品工业中的作用日益显著。但目前乳糖酶并未在中国食品和乳制品工业中得到广泛应用，主要原因是：(1)乳糖酶的单位产量较低；(2)市场上销售的乳糖酶多为胞内酶，酶的耐热性差、适用范围窄；且这种酵母来源(Kluyveromyces lactis、Kluyveromyces fragilis)的胞内酶，需破碎细胞才能得到，提取、纯化工艺繁杂。由于上述等原因致使乳糖酶销售价格过高、应用成本昂贵、适用范围窄，不能满足食品和乳制品工业多方面的需要。
本论文研究的目的是从本实验室保存的一株产乳糖酶的亮白曲霉(Aspetgillus candidus)中克隆并获得拥有自主知识产权的乳糖酶基因，构建高效表达、有效分泌乳糖酶的重组毕赤酵母生物反应器，以解决乳糖酶单位产量低、胞内酶提取困难的问题。期望利用生物技术开发一条乳糖酶高效生产的新途径。
中国农业科学院博士学住论文
结论
1．本研究从一株产乳糖酶的亮白曲霉（ASpergillu candidus冲克隆得到了乳糖
酶基因组DNA及CDN＊序列，序列分析表明，该乳糖酶基因组DNA序列长3458hp，
其中含有8个内含子，CDNA编码区长3015hp，共编码1005个氨基酸，前19个氨
基酸为信号肽序列，氨基酸序列中共含有11个潜在的糖基化位点。将不同来源的
乳糖酶基因与此基因进行比较发现，该基因与绝大多数乳糖酶基因同源性较低；与
商业化应用的、在本研究中作为对照的 A．o叭ae ATCC 20423的乳糖酶基因组 DNA
和衍生的氨基酸序列比较，两基因CDNA的同源性为99％，氨基酸的同源性为
99．5％，二者的基因组DNA序列中有19个碱基不同，内含子区域有5个碱基不同，
CDNA序列中有 14个碱基不同，其中 3个碱基的变化引起了 3个氨基酸的改变：ZI＋，
T从cry a幻一？U．can＆血小 牡0，1U．口W二a一～＊U．can”山小989，NU．O炒二ae
—D（．candidusX但经实验证实，二者在酶学性质上有显著的差异，由此说明我们
克隆到了一个新基因，我们己将该基因在 GENEBANK中登录，登录号为：EMBL
ACCESSIONNO．AJ431643。
2．研究了亮白曲霉的乳糖酶的酶学性质：其最适叫．2，最适温度为60OC，PH
稳定范围在pH3习之间，比活为706．54U／mg，以ONPG为底物时的义为
1石7mmol几，七x为3．33卜mo厂L。将亮白曲霉的乳糖酶与商业化应用的、本研究作
为对照的米曲霉ATCC 20423的乳糖酶进行了酶学性质的比较，结果表明亮白曲霉
的乳糖酶在热稳定性、金属离子稳定性、比活、（、pH范围方面均优于后者，该乳
糖酶具有更优越的酶学性质，在工业化生产中具有更广阔的应用前景。
3．亮白曲霉的乳糖酶基因在大肠杆菌和毕赤酵母中均得到了表达，大肠杆菌
表达的乳糖酶蛋白分子量约为 11 okD，与理论分子量山 okD湘符；毕赤酵母表达的
乳糖酶蛋白分子量为120．130kD，二者分子量之间有差别的原因可能是毕赤酵母表
达系统可对该蛋白进行翻译后的糖基化修饰作用，而原核表达系统不具备该功能。
毕赤酵母表达系统可高效地表达有生物活性的亮白曲霉的乳糖酶，分泌表达量
近 6g／L，酶活为 3600U／InL，且目的蛋白占发酵液中总分泌蛋白的 90％以上，发酵
液中杂蛋白含量很少。因此本研究开辟了一条产量高、分离纯化简便、廉价大规模
生产乳糖酶的新途径。
4．初步进行了亮白曲霉的乳糖酶水解新鲜牛奶的应用效果研究。将不同酶量
的孪糖酶O000U、1500U）分另添力到10mL牛奶中，测定在不同温度下OZ℃、37
OC、50oC、60oCX不同时间内，乳糖酶对牛奶中的乳糖的水解程度。通过高压液?
The enzyme beta~D-galactosidase(beta-D-galactoside galactohydrolase, EC 3.2.1.23, also referred to as lactase)hydrolysis the disaccharide lactose to glucose and galactose. It is widely distributed various microorganisms such as bacteria, fungi, and also in various plants and animals intestine. Lactase can be used for treatment of "lactose intolerance", and production of low-lactose milk, whey and other dairy products. Although lactase has important applications in the fields of medicine, dairy industry, some problems obstructs large-scale industrial production of lactase. Main problems ate: (l)Low production and Enzymatic activity of lactase; (2)Commercial lactase mainly derived from yeasts(Kluyveromyces lactis <. Kluyveromyces fragilis) with low thermostability and narrow pH range; (3)An intracellular lactase resulting in the high cost due to extraction and purification processes.
The purposes of our research are: (1) Screening lactase-yielded strain with superior enzymatic characteristics, identifying and isolating the lactase gene from the microorganism. (2) Enhancing production of lactase and reducing production cost via constructing high effective bioreactor of Pichia pastoris which can express and secrete lactase effectively.
The results obtained as follows:
1. A lactase-yielded strain Aspergillus candidus is screened. Genomic DNA and cDNA sequences of lactase from Aspergillus candidus are cloned. Sequences analysis reveals that the genomic DNA with the length of 345 8bp containing eight introns, cDNA with the length of 3015bp encoding a polypeptide of 1005 amino acid residues. Signal peptide is consisted of 19 amino acid residues. 11 potential N-glycosylation sites are assumed. Comparing the gene cDNA and the deduced amino acid sequences with the lactase sequences from various organism sources shows a very low homology, and the highest homology of cDN A and the deduced amino acid sequence are observed between the lactase of Aspergillus candidus and from Aspergillus oryzae ATCC 20423 which used as control strain. Homology of cDNA sequence is 99%, and homology of the deduced amino acid sequence is 99.5%. Although only 3 amino acid residues are
different in the lactase from A. candidus2, 1(A. oryzae P (A. candidus); 420, l(A. ory~ae)^M(A. candidus); 989, N(^. oryzae-℃D(A. candidus)}, the characteristics of the enzyme exhibits obvious difference from that of A. oryzae. The lactase gene from Aspergillus candidus is a new gene for food and diary industry.
2. The lactase from Aspergillus candidus shows optimum pH of 5.2, optimum temperature of 60℃, and the stable pH ranging from 3.0 to 9.0. Specific activity is 706.54U/mg. The Km is 1.67mmol/L, Fraax is 3.33umol/L with ONPG as a substrate. The enzymatic characteristics of Aspergillus candidus is superior in many aspects to that of Aspergillus oryzae ATCC 20423 including the thermostability, specific activity, metal ion stability, Km, and pH stable range.
3. Lactase gene from Aspergillus candidus is expressed in E. coil and Pichia pastoris. By SDS-PAGE, the molecular weight of lactase expressed in E. coil is estimated to be about 11 OkD according with calculated molecular weight. The molecular weight of lactase protein expressed in Pichia pastoris is about 120-13 OkD probably due to glycosylation. Lactase from Aspergillus candidus is expressed in Pichia pastoris efficiently. High-level secreted lactase is about 6g/L with enzymatic activity of 3600U/mL. Therefore, the research open a new and convenient avenue for high-yielded lactase production.
4. 3000U and 1500U of lactase is added separately in lOmL milk and the lactose content in milk by HPLC at the condition of various temperature (12℃ > 37 ℃ > 50 ℃ .. 60 ℃) and various incubated times is determined. The result shows, (1) lactose hydrolysis rate under the condition of 3000U lactase is higher than that of 1500U at the same temperature and incubated tune. (2) The lactose hydrolysis rate is higher at the higher temperature than that of lower temperature at the same incubated time. The
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