CN106399426B - 一种利用镉米生产海藻糖的方法 - Google Patents
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Abstract
合成序列为SEQ NO1金属亲和多肽BMP并展示在毕赤酵母表面构建BMP基因工程菌用于镉米降镉,合成序列为SEQ NO2的海藻糖合成酶TreS基因并构建基因工程菌用于生产海藻糖合成酶;镉米经磨浆、液化、压滤分离后得镉蛋白和液化糖浆,镉蛋白经蛋白酶分解得蛋白分解液,而液化糖浆加糖化酶后转化为麦芽糖浆;将构建的BMP基因工程菌加入到镉蛋白分解液中搅拌吸镉、离心分离后得吸镉酵母和大米蛋白肽;将该大米蛋白肽用作TerS基因工程菌的发酵培养基主成分,将其发酵扩培生产的菌液均质、破碎即得TreS粗酶液,将其加入到麦芽糖浆中进行催化反应,然后再进行相应处理,即可将镉米充分利用生产海藻糖。
Description
技术领域
本发明属于食品加工和生物技术领域,具体涉及一种利用生物技术构建基因工程菌用于镉超标稻米(简称镉米)降镉加工生产海藻糖的方法。
背景技术
镉米,一般指镉含量超标(中国国标为0.2 mg/kg)的大米。过去几十年,由于片面追求GDP的增长和对环保的不重视,中国土壤污染越来越严重,大米镉等重金属超标也越来越严重;2008年南京农大潘根兴团队在全国多个县级以上市场随机采购样品,结果表明10%左右的市售大米镉超标;2013年广东镉米风波中报道湖南、江西等地流向广东的镉米最高竟超过国标6倍;由于土地污染治理困难,今后相当长一段时间中国将面临大量镉米的加工处理难题;杨居荣等人的研究表明,镉在稻米中主要与蛋白质结合,与淀粉结合较少;而现在的去镉方法多集中在物理或化学方法,少见生物方法去镉,更缺乏将镉米脱镉后加工成高附加值产品如海藻糖等产品的研究。
海藻糖(Trehalose)是由两分子葡萄糖以α,α-1,1-糖苷键相连而成的非还原性二糖,由于其具有对生物体优良的抗逆保护作用等优良性能而被誉为“生命之糖”和“二十一世纪的新型糖类”,其大规模制造方法受到广泛关注;海藻糖现在主要以双酶法或单酶法生产,单酶法以麦芽糖为原料,由于只用一种酶,因而具有简便的优点,但现有海藻糖合成酶存在反应温度偏低,易染杂菌,反应时间较长,转化率偏低的缺陷,而用来生产海藻糖合成酶的基因工程菌常用甘油等为发酵培养基主成分,成本较高,这些缺点均严重影响了单酶法的推广和应用。
展示蛋白或酶的酵母菌细胞具有固定化可重复使用的优点,又具有制备简单,可高密度培养获得大量菌体,成本较低的特点,因而自上世纪末问世以来,发展迅猛,已有许多种蛋白或酶被成功表达的报道;冯侠等(基于展示技术的Cd2+结合肽筛选与酵母重金属吸附研究[J].河南农业科学,2013,42(9):142-145)报道将十二肽展示在酿酒酵母EBY100上,诱导24h后得到能吸附Cd2+的酵母菌,但其对Cd2+的吸附率不高,只有30.4%。
发明内容
本发明所要解决的技术问题一是构建吸附效果更好的酵母基因工程菌来更有效地脱去镉米蛋白酶解游离出来的镉,二是构建能产耐高温、反应时间较短、转化率较高的海藻糖合成酶基因工程菌,三是利用脱镉大米蛋白肽作为海藻糖合成酶基因工程菌的主成分培养基,进一步降低海藻糖合成酶生产成本,从技术上进一步方便生产使用并降低大米生产海藻糖的加工利用成本,提升镉米的加工附加值,降低镉米因不能食用只能贱卖所造成的经济损失。
本发明采取的技术方案是:
(1)将镉米浸泡1~3 h,磨成50~80目的米浆,加水调成20~40%(W/W)浓度的淀粉乳,调pH到6.1~6.5,按5~10U/g加α-淀粉酶在90~110℃条件下分解20~60分钟,然后降温到55~65℃,用板框压滤机将其分离成镉蛋白与液化糖浆;
(2)将上述(1)所得的镉蛋白以重量计加入1~3倍的水,加NaOH调pH到7~8,然后按1~3%(W/W)的比例加入蛋白酶搅拌4~8 h后,按2~6%(W/W)加入展示金属亲和肽BMP的毕赤酵母基因工程菌(简称BMP基因工程菌),搅拌16~24 h吸附蛋白酶分解游离出来的镉,然后用离心机将其分离成吸镉酵母与脱镉大米蛋白肽,将脱镉大米蛋白肽冷冻干燥后用作海藻糖合成酶TreS基因工程菌(简称TreS基因工程菌)发酵培养基的主成分;
(3)在上述(1)所述液化糖浆中按15~25 U/g的量加入β-淀粉酶,糖化2~4h后,得麦芽糖浆;
(4)将TreS基因工程菌按4~8%的接种量接入大米蛋白肽为主成分的培养基中,发酵培养至OD约为0.6时加入终浓度为0.5mmol/L的乳糖作为诱导剂,在22~26℃,200r/min诱导16~24 h,将发酵液经均质机破碎菌体细胞后即得海藻糖合成酶粗酶液;
(5)以重量计将5~10份(4)所述海藻糖合成酶粗酶液加入95~90份(3)所述的麦芽糖浆中,在pH4.5~7,42~52℃温度下进行催化反应2~8 h后过滤分离,将过滤分离后所得的反应液再经压滤、离交、色谱分离,所得的海藻糖浆经浓缩、干燥、结晶即得到结晶海藻糖,而色谱分离所得麦芽糖浆返回合并到(3)所述的麦芽糖浆中作为海藻糖生产原料。
如[0006]所述BMP基因工程菌制备方法如下:通过半理性设计等方法,在前人基因序列基础上进行改进,设计合成一系列新的基因序列,委托生物公司进行序列合成和多个相关基因工程菌构建;然后通过验证实验选择合成镉亲和性好的SEQ NO1序列(见后附序列表SEQ NO1),将合成得到的DNA克隆在pUC57载体上,然后进一步亚克隆到pPIC9K载体中,再转化到毕赤酵母GS115中,得到展示金属亲和肽BMP的毕赤酵母基因工程菌,即BMP基因工程菌,其对镉的吸附率在70~80%。
如[0006]所述TreS基因工程菌的制备方法如下:通过半理性设计等方法,在前人基因序列基础上进行改进,设计合成一系列新的基因序列,委托生物公司进行序列合成和多个相关基因工程菌构建;然后通过验证实验选择合成具有耐高温、催化反应时间较短且活性较高特点的海藻糖合成酶DNA序列SEQ NO2(见后附序列表SEQ NO2),将合成的基因构建到pET28a(+)载体中的Nco I和Xho I酶切位点之间,将构建好的基因转化到BL21(DE3)ply sS大肠杆菌中,得到一种TreS反应温度在42~52℃、反应时间2~8 h的TreS基因工程菌。
如[0006](2)、(4)中所指用作TreS基因工程菌发酵培养基是按每L培养基用(2)中所述冷冻干燥后的脱镉大米蛋白肽20~30 g,与2~3 g MgSO4及5~10 ml微量元素液混合后,再加水复配成1L制成的;其中微量元素液预先按:FeSO4·7H2O 10 g/L、ZnSO4·7H2O 2.25g/L、CuSO4·5H2O 2.25 g/L、MnSO4·5H2O 0.5 g/L、CaSO4·2H2O 2 g/L用1 mol/L HCl溶解配制并储于棕色试剂瓶中备用。
本发明与现有技术相比,脱镉率较高,达到75%左右;用脱镉大米蛋白为主成分培养基培养海藻糖合成酶大肠杆菌基因工程菌比原来用甘油等为主成分的培养基成本降低50%以上;用该菌生产的海藻糖合成酶催化麦芽糖生产海藻糖,转化率达到73.92%左右,反应温度为42~52℃,难染杂菌,且反应时间较短,只要2~8 h,因而海藻糖生产成本大大降低。
附图说明
图1为本发明生产工艺流程图
具体实施方式
以下通过具体实施例对本发明作进一步具体详细描述,但本发明的实施方式不限于此,对于未特别注明的工艺参数,可参照常规技术进行。
实施例1 pPIC9K-BMP载体的构建:将BMP金属亲和肽DNA序列SEQ NO:1人工合成,序列的5’端添加Eco RI限制性内切酶位点,3’端添加Not I位点。Eco RI-BMP-Not I委托生物公司合成并克隆到pUC57载体的多克隆位点上,命名为pUC57-BMP;分别将pUC57-BMP和pPIC9K载体进行Eco RI和Not I双酶切反应,37℃水浴4 h后用1.2%琼脂糖凝胶电泳进行鉴定;采用凝胶回收试剂盒对目的基因片断BMP和pPIC9K载体片段进行回收;再用T4 DNALigase进行连接反应,22℃过夜连接,将其转化到DH5a感受态细胞中,并涂布在含有amp和kana抗性的LB固体培养基(1%胰蛋白胨+0.5%酵母提取物+0.5%NaCl+1.5%琼脂)上37℃过夜培养后挑取克隆进行摇菌培养,质粒抽提,并进行Eco RI和Not I双酶切反应,并用1.2%琼脂糖凝胶电泳鉴定;将阳性克隆命名为pPIC9K-BMP;
实施例2 pPIC9K-BMP转化到毕氏酵母GS115中,按照如下步骤进行:(1)煮沸1ml鲑鱼精DNA 5min,迅速冰浴后以制备单链担体DNA;(2)将pPIC9K-BMP用Sac I内切酶进行线性化处理;(3)将感受态酵母菌GS115离心,用Tips去除残余的LiCl溶液;(4)对于每一个转化,按以下顺序加入: 50% PEG3350,240 ml;1M LiCl,36 ml;2 mg/ml 单链Salmon spermDNA,25 ml;5~10 mg/50 ml H2O 质粒DNA,50 ml;
(5)剧烈旋涡混匀至沉淀菌体完全分布均匀(约1 min);(6)30 ℃水浴孵育30min;(7)42 ℃水浴热休克20~25 min;(8)6000~8000 rpm离心收集酵母菌体;重悬酵母于1 ml YPD培养基(2% Trypton + 2% Dextrose );(9)30℃摇床孵育;(10) 1~4 h后,取25~100 ml 菌液铺YD选择性培养基(1.67% YNB+2 % dextrose+1%琼脂粉)平板,于30℃培养2~3 d鉴定(将表达菌株和对照菌株在固体培养基平板,30℃培养至转化子出现)。
实施例3 BMP基因工程菌制备:将毕赤酵母重组菌接种到YPD液体培养基中,30℃,180 r/min培养24 h进行活化;按1%的接种量转接至新鲜的BMGY培养基(1%Yeast extract+2%Peptone+1.34%YNB+4×10-5 生物素+1%甘油/0.5%甲醇+10% 1M PBS)中,30℃,180 r/min培养4~5 d,每24 h添加甲醇进行诱导;收集发酵菌体,菌泥用pH8.0缓冲液洗涤3次;经离心后得湿菌体,制得BMP基因工程菌。
实施例4 BMP基因工程菌对镉米脱镉处理:将镉米浸泡2 h,磨成60目的米浆,加水调成30%(W/W)浓度的淀粉乳,调pH到6.3,按8 U/g加α-淀粉酶在100℃条件下分解30~40分钟,然后降温到60℃,用板框压滤机将其分离成镉蛋白与液化糖浆;将所得镉蛋白以重量计加入2倍的水,加NaOH调pH到7.5,然后按2%(W/W)的比例加入蛋白酶搅拌6 h后,按4%(W/W)加入BMP基因工程菌,振荡20 h吸附蛋白酶分解游离出来的镉等重金属,然后用离心机将其分离成吸镉酵母与脱镉大米蛋白肽,将脱镉大米蛋白肽冷冻干燥后用作海藻糖合成酶基因工程菌发酵培养基的主成分。
实施例5 酵母工程菌脱镉程度的测定:按国标法用原子吸收分光光度计测定Cd2+离子质量浓度,然后计算吸收率,进而计算对镉的吸附率,测定结果如下:
表1 BMP酵母工程菌脱镉效果
镉蛋白中镉含量 | 脱镉多肽中镉含量 | BMP基因工程菌脱镉率 |
0.60±0.11mg/kg | 0.15±0.03mg/kg | 75.0±5.0% |
实施例6 TreS基因工程菌的制备:按SEQ NO2所示TreS DNA序列,将合成的基因构建到pET28a(+)载体中的Nco I和Xho I酶切位点之间,再将构建好的基因转化到BL21(DE3)ply sS大肠杆菌中,得到一种反应温度在42~52℃、反应时间2~8 h的TreS基因工程菌。
实施例7 TreS基因工程菌发酵培养基的配制:每L培养基按将上述所得冷冻干燥后的脱镉大米蛋白肽以重量计按25 g 加入2.08 g MgSO4、10 ml微量元素液的比例再加水复配成1L发酵培养基方式配制,供TreS基因工程菌经LB种子发酵和摇瓶发酵后进一步发酵扩培生产用;其中微量元素液按:FeSO4·7H2O 10 g/L、ZnSO4·7H2O 2.25 g/L、CuSO4·5H2O2.25 g/L、MnSO4·5H2O 0.5 g/L、CaSO4·2H2O 2 g/L用1 mol/L HCl溶解并储于棕色试剂瓶中配制备用。
实施例8 海藻糖合成酶的制备:以[0019]中所述培养基,将TreS基因工程菌按5%的接种量接入,培养至OD约为0.6时加入终浓度为0.5mmol/L的乳糖作为诱导剂,在25℃,200r/min诱导20 h,将发酵液经均质机破碎后离心,取上清液即为海藻糖合成酶粗酶液。
实施例9 海藻糖浆的制备:在[0016]所述液化糖浆中按20 U/g的量加入β-淀粉酶,糖化3 h后,得麦芽糖浆;以重量计将10份[0020]中所述的海藻糖合成酶粗酶液加到90份该麦芽糖浆中,在pH4.5~7,42~52℃温度下进行催化反应6 h后,用HPLC对反应产物进行各种糖含量分析,结果为海藻糖21.51±2.33%,麦芽糖5.58±1.37%,葡萄糖含量1.83±0.36%,由底物浓度29.10%,计算转化率为21.51/29.1=73.92%;将催化反应所得糖浆再经压滤、离交、色谱分离,即得海藻糖浆和麦芽糖浆,如图1所示,海藻糖浆可根据市场需要调配成海藻糖浓度为20~70%的糖浆出售或进一步制成结晶海藻糖,而麦芽糖浆可重新用作海藻糖生产原料。
实施例9 结晶海藻糖的生产:[0021]所述生产所得海藻糖浆如图1所示经浓缩、结晶、离心、干燥后得到纯度在98%以上的结晶海藻糖。
Claims (2)
1.一种利用镉米生产海藻糖的方法,其特征在于,所述方法包括如下步骤:
(1)将镉米浸泡1~3 h,磨成50~80目的米浆,加水调成20~40%(W/W)浓度的淀粉乳,调pH到6.1~6.5,按5~10U/g加α-淀粉酶在90~110℃条件下分解20~60分钟,然后降温到55~65℃,用板框压滤机将其分离成镉蛋白与液化糖浆;
(2)将上述(1)所得的镉蛋白以重量计加入1~3倍的水,加NaOH调pH到7~8,然后按1~3%(W/W)的比例加入蛋白酶搅拌4~8 h后,按2~6%(W/W)加入展示金属亲和肽BMP的毕赤酵母基因工程菌(简称BMP基因工程菌),振荡16~24 h吸附蛋白酶分解游离出来的镉,然后用离心机将其分离成吸镉酵母与脱镉大米蛋白肽,将脱镉大米蛋白肽冷冻干燥后用作海藻糖合成酶TreS基因工程菌(简称TreS基因工程菌)发酵培养基的主成分;
(3)在上述(1)所述液化糖浆中按15~25 U/g的量加入β-淀粉酶,糖化2~4h后,得麦芽糖浆;
(4)将TreS基因工程菌按4~8%的接种量接入大米蛋白肽为主成分的培养基中,发酵培养至OD约为0.6时加入终浓度为0.5mmol/L的乳糖作为诱导剂,在22~26℃,200r/min诱导16~24 h,将发酵液经均质机破碎即为海藻糖合成酶粗酶液;
(5)以重量计将5~10份(4)所述海藻糖合成酶粗酶液加入95~90份(3)所述的麦芽糖浆中,在pH4.5~7,42~52℃温度下进行催化反应2~8 h后过滤分离,将过滤分离后所得的反应液再经压滤、离交、色谱分离,所得的海藻糖浆经浓缩、干燥、结晶即得到结晶海藻糖,而色谱分离所得麦芽糖浆返回合并到(3)所述的麦芽糖浆中作为海藻糖生产原料;
其中(2)中所述金属亲和肽BMP的DNA编码序列如SEQ NO.1所示,所述BMP基因工程菌制备方法如下:合成BMP的DNA序列,将合成得到的DNA克隆在pUC57载体上,然后进一步亚克隆到pPIC9K载体中,再转化到毕赤酵母GS115中,得到BMP基因工程菌,其对镉的吸附率在70~80%;
其中(2)、(4)中所述海藻糖合成酶TreS的DNA编码序列如SEQ NO.2所示,所述TreS基因工程菌的制备方法如下:合成TreS的DNA序列,将合成的基因构建到pET28a(+)载体中的NcoI和Xho I酶切位点之间,将构建好的基因转化到BL21(DE3) ply sS大肠杆菌中,得到一种TreS反应温度在42~52℃、反应时间2~8 h的TreS基因工程菌。
2.如权利要求1所述的方法,其特征在于,其中(2)、(4)中所指用作TreS基因工程菌发酵培养基是按每L培养基用(2)中所述冷冻干燥后的脱镉大米蛋白肽20~30 g,与2~3 gMgSO4及5~10 ml微量元素液混合后,再加水复配成1L制成的;其中微量元素液预先按:FeSO4·7H2O 10 g/L、ZnSO4·7H2O 2.25 g/L、CuSO4·5H2O 2.25 g/L、MnSO4·5H2O 0.5 g/L、CaSO4·2H2O 2 g/L用1 mol/L HCl溶解配制并储于棕色试剂瓶中备用。
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