CN114540380A - 一种山梨醇脱氢酶sorDHGo基因及编码蛋白和在制备玻色因中的应用 - Google Patents
一种山梨醇脱氢酶sorDHGo基因及编码蛋白和在制备玻色因中的应用 Download PDFInfo
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- CN114540380A CN114540380A CN202210265361.3A CN202210265361A CN114540380A CN 114540380 A CN114540380 A CN 114540380A CN 202210265361 A CN202210265361 A CN 202210265361A CN 114540380 A CN114540380 A CN 114540380A
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Abstract
本发明公开了一种山梨醇脱氢酶sorDHGo基因及编码蛋白和在制备玻色因中的应用。该sorDHGo基因的核苷酸序列如SEQ ID NO.1所示,其编码的蛋白的氨基酸序列如SEQ ID NO.2所示。本发明中β‑丙酮木糖苷可立体专一性地还原得到玻色因,还避免使用污染、危险的化合试剂,可实现玻色因绿色生产制造,克服了现有路线中工艺放大困难、危险废弃物多等缺点,同时大大降低了生产成本。
Description
技术领域
本发明属于生物技术领域,具体涉及一种山梨醇脱氢酶sorDHGo基因及编码蛋白和在制备玻色因中的应用。
背景技术
玻色因(Pro-XylaneTM),化学名为羟丙基四氢吡喃三醇(CAS:439685-79-7),是一种具有抗衰老活性的木糖衍生物,常用于化妆品中。玻色因(Pro-XylaneTM)于2006年9月成功上市。研究表明玻色因具有广泛的生物活性,玻色因可直接影响皮肤三层结构中的细胞外基质,可以激活或促进皮肤中黏多糖(GAGs)的合成,改善真皮与表皮间的黏合度,促进受损组织的再生,帮助维持真皮的弹性,可有效地保持皮肤紧致、细腻,延缓皮肤的衰老。此外,玻色因还可促进透明质酸和胶原蛋白生成。玻色因易于生物降解,不会在生物体内积累,没有毒性。目前,生产玻色因的方法主要有两种,一种是化学合成法。另一种方法是生物合成方法。
国外的化学合成法是以木糖为原料,在碱性条件下,与2,4-戊二酮缩合反应12h,经强酸性阳离子交换树脂酸化,再用硼氢化物对羰基还原反应12h,合成玻色因。还有的方式也是以木糖为原料,在碳酸氢钠作用下,与2,4-戊二酮进行缩合反应,转换为C-糖苷,再用重金属催化剂Ru/C对羰基还原,合成玻色因。该合成方法产率高,但NaOH溶液腐蚀性大,对反应器材质的要求高,对环境有污染,所产生三废治理花费高;此外,该法还存在还原选择性低,使用硼氢化钠造成污染,且产物难以提纯等缺点。
国内在此路线上进行了改进,第一步是以木糖为原料,采用强碱性阴离子树脂代替了普通的无机碱,第二步再用硼氢化钠对羰基进行还原,合成玻色因。但由于这两步反应的产物均采用了柱层析纯化,操作复杂,工业化放大有一定难度。因此,需要开发一种低能耗、绿色环保的生物酶合成法。
发明内容
针对现有技术中的上述不足,本发明提供一种山梨醇脱氢酶sorDHGo基因及编码蛋白和在制备玻色因中的应用,本发明不但可以立体专一性地还原生成了玻色因,还避免了使用污染、危险的化学试剂,同时使得产品分离提纯简单高效。该方法能够高效地提高合成玻色因,具有见效快、成本低、操作简单、方便实施、产品无毒无残留等特征,以满足玻色因的大规模生产。
为实现上述目的,本发明解决其技术问题所采用的技术方案是:
一种山梨醇脱氢酶sorDHGo基因,该sorDHGo基因的核苷酸序列如SEQ ID NO.1所示。
进一步地,蛋白的氨基酸序列如SEQ ID NO.2所示。
进一步地,sorDHGo基因的核苷酸序列还可以为与SEQ ID NO.1所示序列具有70%以上同源性,且编码具有相同功能蛋白的序列。
上述山梨醇脱氢酶sorDHGo基因来源于保藏号为CGMCC 1.565的Gluconobacteroxydans(氧化葡糖杆菌),该菌株购自中国微生物菌种保藏中心。
上述山梨醇脱氢酶sorDHGo基因,或蛋白在制备玻色因中的应用。
进一步地,包括以下步骤:
(1)将乙酰丙酮、木糖与催化剂在溶剂中混合加热反应,得到β-丙酮木糖苷;
(2)将β-丙酮木糖苷、权利要求1、3~5任一项所述的山梨醇脱氢酶sorDHGo基因表达的β-丙酮木糖苷还原酶、辅酶与异丙醇或葡萄糖脱氢酶GDH在缓冲液中混合反应,即可制得玻色因。
进一步地,催化剂为氢氧化钠、氢氧化锂或碳酸氢钠。
进一步地,催化剂为氢氧化钠。
进一步地,步骤(1)中反应温度为25~100℃,反应时间为0~24h。
进一步地,反应温度为50℃,反应时间为0.75h。
进一步地,步骤(2)中辅酶选自氧化型β-烟酰胺腺嘌呤二核苷酸磷酸四钠(NADP+)、氧化型烟酰胺腺嘌呤二核苷酸二钠(NAD+)、还原型β-烟酰胺腺嘌呤二核苷酸磷酸四钠(NADPH)、还原型烟酰胺腺嘌呤二核苷酸二钠(NADH),优选还原型β-烟酰胺腺嘌呤二核苷酸磷酸四钠(NADPH)或还原型烟酰胺腺嘌呤二核苷酸二钠(NADH)。
进一步地,步骤(2)中异丙醇与β-丙酮木糖苷的比例为(1~1.5)mL:1g;葡萄糖脱氢酶GDH与β-丙酮木糖苷的比例为200U/g。
进一步地,步骤(2)中反应温度为25℃~40℃;反应时间为2~72h。
本发明的有益效果:
本发明中β-丙酮木糖苷可立体专一性地还原得到玻色因,还避免使用污染、危险的化合试剂,可实现玻色因绿色生产制造,克服了现有路线中工艺放大困难、危险废弃物多等缺点,同时大大降低了生产成本。
附图说明
图1为重组质粒pET28a-sorDHGo图谱;
图2为重组β-丙酮木糖苷还原酶蛋白表达SDS-PAGE图。
具体实施方式
下面对本发明的具体实施方式进行描述,以便于本技术领域的技术人员理解本发明,但应该清楚,本发明不限于具体实施方式的范围,对本技术领域的普通技术人员来讲,只要各种变化在所附的权利要求限定和确定的本发明的精神和范围内,这些变化是显而易见的,一切利用本发明构思的发明创造均在保护之列。
实施例1不同催化剂对β-丙酮木糖苷制备反应的影响
木糖(10g)用去离子水(40mL)完全溶解,加入到100mL的三口瓶中,加入不同的催化剂(1.25eq),并在90℃反应18小时,化学反应式如下,反应结果如表1所示。
表1催化剂对β-丙酮木糖苷制备的影响
碱 | 反应温度℃ | 反应时间h | 产率% |
NaHCO<sub>3</sub> | 90 | 18h | 87% |
LiOH | 90 | 18h | 56% |
NaOH | 90 | 18h | 88% |
根据表1结果可知,上述催化剂下,本发明方法均能获得较高产率。综合来看,催化剂为NaOH与NaHCO3反应效果较佳。
实施例1反应温度及时间对β-丙酮木糖苷制备反应的影响
木糖(10g)用去离子水(40mL)完全溶解,加入到100mL的三口瓶中,NaOH(3.32g,1.25eq),并在50-90℃反应0-18小时,化学反应式如下,反应结果如表2所示。
表2反应温度及时间对β-丙酮木糖苷制备的影响
碱 | 反应温度℃ | 反应时间h | 产率% |
NaOH | 90 | 18h | 86% |
NaOH | 90 | 1h | 89% |
NaOH | 50 | 0.75h | 97.5% |
NaOH | 25 | 0.75h | 12% |
根据表2的结果可知,上述催化剂、温度和反应时间下,本发明方法均能得到目标产物β-丙酮木糖苷。综合来看,催化剂为NaOH,反应温度为50℃,反应时间为0.75h时,反应效果较佳。
实施例3玻色因的制备
1、β-丙酮木糖苷的制备
木糖(100g)用去离子水(400mL)完全溶解,加入到1000mL的三口瓶中,NaOH(53.2g,2eq),并在50℃反应1小时,结束后,优选采用4mol/L盐酸将反应溶液的pH值调节至中性,除去溶剂,得到第一中间产物β-丙酮木糖苷。
2、β-丙酮木糖苷还原酶基因克隆与重组菌构建
本发明所需的酶都是通过公司合成相应基因后构建在表达质粒上再通过大肠杆菌发酵生产制得;其具体包含以下步骤:
来源于Gluconobacter oxydans CGMCC 1.565(购自中国微生物菌种保藏中心)的山梨醇脱氢酶sorDHGo基因,基因序列如SEQ ID NO.1所示,氨基酸序列如SEQ ID NO.2所示;重组质粒如图1所示,经过密码子序列优化后,将序列送到金开瑞生物公司进行全基因合成,采用EcoRI/XhoI酶进行双酶切后亚克隆到pET28a表达载体上。
确认序列正确的质粒转入E.coli BL21(DE3)pLysS(擎科生物)感受态细胞进行平板培养,获得阳性单克隆菌株;重组阳性克隆菌株β-丙酮木糖苷还原酶蛋白表达情况如图2所示。
3、β-丙酮木糖苷还原酶(sorDHGo)蛋白表达
单菌落转入5mL含50μg/mL卡那霉素和10μg/mL氯霉素的LB培养液中(37℃)进行培养,当细胞生长至对数期后接种到500mL含50μg/mL卡那霉素和10μg/mL氯霉素的LB培养液中,同样生长到对数期时转入30L培养发酵罐里进行培养并进行最终的蛋白表达,30L发酵罐中蛋白表达发酵培养基构成为:10g/L胰蛋白胨、5g/L酵母粉,5g/L NaCl,5g/L磷酸氢二钾、5g/L磷酸氢二钾以及10%的甘油。
在30L发酵罐培养中,当细胞OD600~2时加入0.5mM异丙基-β-D-硫代吡喃半乳糖苷(IPTG)16℃诱导蛋白表达16小时,最后高速离心收集细胞(4000rpm,20min)获得酶过量表达湿细胞80~100g。
菌体细胞用磷酸盐缓冲液(K2HPO4-KH2PO4)缓冲液(50mM,pH 8.0)在冰盆上混合均匀,彻底悬浮;然后进行低温高压破碎细胞壁,高速离心(16000rpm,45min)除细胞壁后获得含酶清液备用。
4、β-丙酮木糖苷还原酶的纯化
含酶清液利用镍柱亲和层析进行蛋白纯化。以下为缓冲液配方:
A液:KPB缓冲液((20mM,pH 7.2),含有10mM咪唑、0.5M NaCl;
B液:KPB缓冲液(20mM,pH 7.2),含有0.5M咪唑、0.5M NaC1;C液:KPB缓冲液(25mM,pH 7.2),150mM NaCl,1mM DTT。
将β-丙酮木糖苷还原酶粗酶液上样到镍柱上,首先用A液洗脱杂蛋白,随后用B液洗脱目标蛋白,洗脱液超滤浓缩到一定体积后用C液进行置换,降低蛋白溶液中的咪唑浓度。根据SDS-PAGE检测的结果收集纯化的蛋白质,加入终浓度为15%(w/v)的甘油,于-80℃保存备用。
5、酶催化还原β-丙酮木糖苷制备玻色因
将35gβ丙酮木糖苷粗品溶解于1.0L磷酸盐缓冲液(K2HPO4-KH2PO4)缓冲液(50mM,pH 8.0)溶液中,再加入2.5g,3.4mM还原型β-烟酰胺腺嘌呤二核苷酸二钠磷酸四钠(NADPH),以及40mL异丙醇。恒温30℃加入粗酶液(sorDHGo),30℃缓慢搅拌反应24小时,反应结束后,离心,上清液纳滤除盐,减压蒸除水分,纯化得34.4g产物,纯度99.6%,产率97.2%。
反应式如下:
采用高效液相色谱法(HPLC)测定上述玻色因含量,具体方法如下:
样品处理:反应液,离心,收集上清,用0.22μm的滤膜过滤后,进行HPLC检测。
色谱柱:ChromCore HICIL-amide 5μm(4.6×250mm);
流动相:A相(水)和B相(乙腈);
等度洗脱:10%A,90%B;
流速:1mL/min;
检测器:CAD检测器(Charged Aerosol Detector)(电喷雾检测器,Thermo FisherScientific);
进样量:10μL。
序列表
<110> 成都格纯生物医药有限公司
<120> 一种山梨醇脱氢酶sorDHGo基因及编码蛋白和在制备玻色因中的应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1455
<212> DNA
<213> 氧化葡糖杆菌(Gluconobacter oxydans)
<400> 1
atgattacgc gcgaaaccct taagtctctt cctgccaatg tccaggctcc cccctatgac 60
atcgacggga tcaagcctgg gatcgtgcat ttcggtgtag gtaacttttt tcgagcccat 120
gaggcgttct acgtcgagca gattcttgaa cacgctccgg actgggcgat tgttggtgtt 180
ggcctgacgg gcagtgaccg ttcaaagaaa aaagccgagg aattcaaggc ccaggactgc 240
ctgtattccc tgaccgagac ggctccgtcc ggcaagagca cggtgcgcgt catgggcgcg 300
ctgcgtgact atctgcttgc cccggccgat ccggaagccg tgctgaagca tcttgttgat 360
ccggccatcc gcatcgtttc catgacgatc acggaaggcg gctacaacat caacgagacg 420
accggtgcgt tcgatctgga gaatgcggca gtaaaggccg acctcaagaa cccggaaaag 480
ccgtctaccg ttttcggtta cgtggtcgag gccctgcgtc gtcgttggga tgccggtggt 540
aaggcattta cggtcatgtc ctgtgataac ctgcgtcata acggcaatgt cgcccgcaag 600
gccttcctcg gctatgcgaa ggcgcgcgat ccggagttgg cgaagtggat tgaggaaaac 660
gcgaccttcc cgaacggaat ggttgatcgc atcaccccga ccgtttcggc ggaaatcgcc 720
aagaagctca acgcggccag tgggctggat gacgacctgc cgctggtggc cgaggatttc 780
catcagtggg tgctggaaga ccagtttgcg gatggccgtc cgccgcttga aaaagccggc 840
gtgcagatgg tcggggacgt gacggactgg gagtacgtca agatccgaat gctcaatgca 900
gggcatgtca tgctctgctt cccaggcatt ctggtcggct atgagaatgt ggatgacgcc 960
attgaagaca gcgaactcct tggcaatctg aagaactatc tcaacaagga tgtcatcccg 1020
accctgaagg cgccttcagg catgacgctc gaaggctatc gggacagcgt catcagccgt 1080
ttctccaaca aggcgatgtc ggaccagacg ctccggattg ctagcgatgg ctgttccaag 1140
gttcaggtgt tctggacgga aaccgtgcgt cgggcgatcg aagacaagcg ggacctgtca 1200
cgtatagcgt tcggaattgc atcctatctc gaaatgctgc gtggtcgcga cgagaagggc 1260
gggacgtatg aatcgtccga gccgacttat ggcgacgccg aatggaagtt ggccaaggcg 1320
gacgacttcg aaagctctct gaagctcccg gcgttcgatg ggtggcgcga tctggatacg 1380
tccgaactgg atcaaaaggt catcgtgctg cggaagatca tccgcgaaaa gggcgtaaaa 1440
gccgccatcc cggcc 1455
<210> 2
<211> 485
<212> PRT
<213> 氧化葡糖杆菌(Gluconobacter oxydans)
<400> 2
Met Ile Thr Arg Glu Thr Leu Lys Ser Leu Pro Ala Asn Val Gln Ala
1 5 10 15
Pro Pro Tyr Asp Ile Asp Gly Ile Lys Pro Gly Ile Val His Phe Gly
20 25 30
Val Gly Asn Phe Phe Arg Ala His Glu Ala Phe Tyr Val Glu Gln Ile
35 40 45
Leu Glu His Ala Pro Asp Trp Ala Ile Val Gly Val Gly Leu Thr Gly
50 55 60
Ser Asp Arg Ser Lys Lys Lys Ala Glu Glu Phe Lys Ala Gln Asp Cys
65 70 75 80
Leu Tyr Ser Leu Thr Glu Thr Ala Pro Ser Gly Lys Ser Thr Val Arg
85 90 95
Val Met Gly Ala Leu Arg Asp Tyr Leu Leu Ala Pro Ala Asp Pro Glu
100 105 110
Ala Val Leu Lys His Leu Val Asp Pro Ala Ile Arg Ile Val Ser Met
115 120 125
Thr Ile Thr Glu Gly Gly Tyr Asn Ile Asn Glu Thr Thr Gly Ala Phe
130 135 140
Asp Leu Glu Asn Ala Ala Val Lys Ala Asp Leu Lys Asn Pro Glu Lys
145 150 155 160
Pro Ser Thr Val Phe Gly Tyr Val Val Glu Ala Leu Arg Arg Arg Trp
165 170 175
Asp Ala Gly Gly Lys Ala Phe Thr Val Met Ser Cys Asp Asn Leu Arg
180 185 190
His Asn Gly Asn Val Ala Arg Lys Ala Phe Leu Gly Tyr Ala Lys Ala
195 200 205
Arg Asp Pro Glu Leu Ala Lys Trp Ile Glu Glu Asn Ala Thr Phe Pro
210 215 220
Asn Gly Met Val Asp Arg Ile Thr Pro Thr Val Ser Ala Glu Ile Ala
225 230 235 240
Lys Lys Leu Asn Ala Ala Ser Gly Leu Asp Asp Asp Leu Pro Leu Val
245 250 255
Ala Glu Asp Phe His Gln Trp Val Leu Glu Asp Gln Phe Ala Asp Gly
260 265 270
Arg Pro Pro Leu Glu Lys Ala Gly Val Gln Met Val Gly Asp Val Thr
275 280 285
Asp Trp Glu Tyr Val Lys Ile Arg Met Leu Asn Ala Gly His Val Met
290 295 300
Leu Cys Phe Pro Gly Ile Leu Val Gly Tyr Glu Asn Val Asp Asp Ala
305 310 315 320
Ile Glu Asp Ser Glu Leu Leu Gly Asn Leu Lys Asn Tyr Leu Asn Lys
325 330 335
Asp Val Ile Pro Thr Leu Lys Ala Pro Ser Gly Met Thr Leu Glu Gly
340 345 350
Tyr Arg Asp Ser Val Ile Ser Arg Phe Ser Asn Lys Ala Met Ser Asp
355 360 365
Gln Thr Leu Arg Ile Ala Ser Asp Gly Cys Ser Lys Val Gln Val Phe
370 375 380
Trp Thr Glu Thr Val Arg Arg Ala Ile Glu Asp Lys Arg Asp Leu Ser
385 390 395 400
Arg Ile Ala Phe Gly Ile Ala Ser Tyr Leu Glu Met Leu Arg Gly Arg
405 410 415
Asp Glu Lys Gly Gly Thr Tyr Glu Ser Ser Glu Pro Thr Tyr Gly Asp
420 425 430
Ala Glu Trp Lys Leu Ala Lys Ala Asp Asp Phe Glu Ser Ser Leu Lys
435 440 445
Leu Pro Ala Phe Asp Gly Trp Arg Asp Leu Asp Thr Ser Glu Leu Asp
450 455 460
Gln Lys Val Ile Val Leu Arg Lys Ile Ile Arg Glu Lys Gly Val Lys
465 470 475 480
Ala Ala Ile Pro Ala
485
Claims (10)
1.一种山梨醇脱氢酶sorDHGo基因,其特征在于,所述sorDHGo基因的核苷酸序列如SEQID NO.1所示。
2.权利要求1所述sorDHGo基因编码的蛋白,其特征在于,所述蛋白的氨基酸序列如SEQID NO.2所示。
3.根据权利要求1所述的山梨醇脱氢酶sorDHGo基因,其特征在于,所述sorDHGo基因的核苷酸序列为与SEQ ID NO.1所示序列具有70%以上同源性,且编码具有相同功能蛋白的序列。
4.根据权利要求1所述的山梨醇脱氢酶sorDHGo基因,其特征在于,所述sorDHGo基因来源于保藏号为CGMCC 1.565的Gluconobacter oxydans。
5.权利要求1所述的山梨醇脱氢酶sorDHGo基因,或权利要求2所述蛋白在制备玻色因中的应用。
6.根据权利要求5所述的应用,其特征在于,包括以下步骤:
(1)将乙酰丙酮、木糖与催化剂在溶剂中混合加热反应,得到β-丙酮木糖苷;
(2)将β-丙酮木糖苷、权利要求1所述的山梨醇脱氢酶sorDHGo基因表达的β-丙酮木糖苷还原酶、辅酶与异丙醇或葡萄糖脱氢酶GDH在缓冲液中混合反应,即可制得玻色因。
7.根据权利要求6所述的应用,其特征在于,所述催化剂为氢氧化钠、氢氧化锂和碳酸氢钠中的至少一种。
8.根据权利要求6所述的应用,其特征在于,步骤(1)中反应温度为25~100℃,反应时间为0~24h。
9.根据权利要求6所述的应用,其特征在于,步骤(2)中所述辅酶为氧化型β-烟酰胺腺嘌呤二核苷酸磷酸四钠、氧化型烟酰胺腺嘌呤二核苷酸二钠、还原型β-烟酰胺腺嘌呤二核苷酸磷酸四钠、还原型烟酰胺腺嘌呤二核苷酸二钠中的至少一种。
10.根据权利要求6所述的应用,其特征在于,步骤(2)中所述反应温度为25℃~40℃;反应时间为2~72h。
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