纤维二糖磷酸化酶表达条件的优化及其酶学性质研究

纤维二糖磷酸化酶(cellobiose phosphorylase,CBP)磷酸化裂解纤维二糖为葡萄糖-1-磷酸,它是木质纤维素转化淀粉的关键酶。该研究将来源于Clostridium thermocellum YM4的cbp基因通过表达载体pET-28a(+)构建重组菌Rosetta(DE3)/pET-28a-CBP用于表达CBP。对其诱导表达条件进行优化,结果显示当诱导温度、异丙基硫代-β-D-半乳糖苷(isopropylthio-β-D-galactoside,IPTG)浓度和诱导时间分别为15℃、0.2 mmol/L和12 h时CBP酶活力最高,为0.47 IU/mL。进一步利用Ni柱分离纯化出带有His标签的CBP,结果表明该重组酶分子质量为95 kDa,比酶活1.03 IU/mg;最适反应温度60℃,最适反应pH 6,半失活时间为60 min;金属离子Cu^(2+)显示出对该酶有强烈的抑制作用,然后依次是Zn^(2+)、Fe^(3+)、Ca^(2+)、Mn^(4+)(P<0.05);而K^(+)、Mg^(2+)和Na^(+)对该酶活力无显著性影响(P>0.05);最后对重组CBP的动力学参数进行了表征,K_(m)值为19.61μmol/mL,V_(max)值为1.19μmol/(mL·min)且K_(cat)值为21.38 s^(-1)。

海栖热袍菌极耐热纤维二糖磷酸化酶在大肠杆菌中的克隆和表达

将来源于极端嗜热菌属海栖热袍菌(Thermotoga maritima)的编码纤维二糖磷酸化酶的结构基因cepA与新型热激质粒pHsh连接,得到重组质粒pHsh-cepA。将重组质粒pHsh-cepA转入大肠杆菌JM109进行表达。SDS-PAGE结果显示,该重组酶的分子量为90kD,与理论值相符。基于热激载体pHsh的重组表达系统具有诱导表达简便、诱导方式廉价的优点,且重组酶热稳定性非常好。这对该酶的大规模发酵应用具有重要意义。

牛瘤胃产纤维二糖磷酸化酶细菌的筛选及产酶条件研究

为了获得产新型纤维二糖磷酸化酶(CBP)的菌株,利用选择性培养基从牛瘤胃液中逐步分离纯化,筛选能以纤维二糖为唯一碳源生长的菌株,分别检测所筛选菌株的β-葡萄糖苷酶(βG)和CBP活力,并对其纤维二糖的利用方式(水解途径或磷解途径)进行鉴定,获得产CBP的菌株,命名为BY-a。采用形态学观察、生理生化试验,结合16S r DNA序列分析,鉴定菌株BY-a为克雷伯氏菌属(Klebsiella)。对菌种产CBP的温度、时间、最适装液量和溶氧量进行初步优化,结果显示,BY-a菌株在装液量为30mL(250mL)、37℃振荡培养24 h时,产CBP活力最高。

二糖磷酸化酶及其在体外合成生物学中的应用

二糖磷酸化酶是以二糖为底物,催化磷酸向糖基转移,生成糖1-磷酸的一类酶。根据催化反应的立体化学过程可以分为保留型和翻转型,即供体底物二糖的异头构型可以在糖1-磷酸产物中保留或翻转。目前已发现的超过10种的二糖磷酸化酶均分布在糖苷水解酶和糖苷转移酶家族中。根据二糖磷酸化酶的功能设计体外生物合成体系,不仅可以用于制备高价值化学品,例如直链淀粉、肌醇、昆布二糖等,还可生产一些大宗产品,例如在氢能和酶燃料电池开发等方面已取得了进展。随着蛋白质工程的不断发展,对二糖磷酸化酶的研究越来越深入,相信未来二糖磷酸化酶将在体外生物合成平台扮演更重要的角色。

乳二糖磷酸化酶的表达优化

为了提高目的蛋白的产量和活性,将克隆自Bifidobacterium longum JCM1217的乳二糖磷酸化酶(LNBP)的基因构建到大肠杆菌BL21中,优化其表达条件。结果显示,诱导时间、诱导温度及IPTG浓度对LNBP的表达水平影响显著。经正交试验,优化出的重组菌表达LNBP的最佳诱导条件为诱导温度30℃, IPTG浓度0.5 mmol/L,诱导时间18 h。在此条件下获得的LNBP比活力达到15.18 U/mg。

纤维二糖磷酸化酶和纤维寡糖磷酸化酶的研究与应用

自然界中一些厌氧的纤维素降解菌能够产生纤维二糖磷酸化酶(Cellobiose Phosphorylase,CBP)和纤维寡糖磷酸化酶(CellodextrinPhosphorylase,CDP)磷酸化裂解纤维二糖和纤维寡糖。CBP和CDP属于糖苷水解酶94家族(Glycoside Hydrolase Family 94,GH94),对β-1,4糖苷键高度专一。目前已广泛研究了不同来源的CBP和CDP的功能性质和催化机理,并通过蛋白质晶体结构分析揭示了二者采用不同聚合度纤维寡糖作为底物的结构基础。因CBP和CDP可催化独特的磷酸化裂解反应和逆向合成反应,已有多篇报道显示CBP和CDP在生产实践上的应用,主要在构建直接利用纤维寡糖的工程酵母菌、构建酶法纤维素转淀粉体系和酶法合成特殊糖类等3个方面。由于对CBP和CDP的持续关注,在此对二者的相关研究进行综述,并提出今后的研究重点。

以纤维二糖为底物利用重组大肠杆菌合成海藻糖

探讨以纤维二糖为底物合成海藻糖的可行性。首先克隆来自施氏假单胞菌A1501的otsA/otsB基因,外源导入Escherichia coli BL21(DE3)构建以葡萄糖为底物合成海藻糖的OtsAB途径。继而通过过表达E. coli本身的galU基因增加海藻糖合成前体物质尿苷二磷酸(uridine diphosphate,UDP)-葡萄糖的含量,使海藻糖产量提高了3倍。通过添加井冈霉素抑制海藻糖的降解,进一步提高海藻糖的产量。在此基础上,过表达来自天然纤维素分解细菌Saccharophagusdegradans的纤维二糖磷酸化酶基因cepA,使重组大肠杆菌具有利用纤维二糖产海藻糖的能力。利用该重组大肠杆菌全细胞催化生产海藻糖,底物为20 g/L纤维二糖,并添加0.05 mmol/L的井冈霉素抑制海藻糖降解时,48 h后可生成1.3 g/L的海藻糖。本研究利用重组大肠杆菌以纤维二糖为底物合成海藻糖,证实了以纤维二糖为底物合成海藻糖的可行性,为纤维二糖来源精细化学品的生产提供了新的思路。

两种耐热磷酸化酶的构建及高效催化合成红景天苷

目的:使用表达耐热蔗糖磷酸化酶的大肠杆菌重组工程菌E. coli BL21/pET-Spase和耐热纤维二糖磷酸化酶的大肠杆菌重组工程菌E. coli BL21/pET-Cpase,发酵培养后粗酶液作为催化剂,以价格低廉的蔗糖为原料合成红景天苷。方法:分别构建耐热蔗糖磷酸化酶和耐热纤维二糖磷酸化酶大肠杆菌重组菌,然后将重组菌、蔗糖、酪醇和磷酸混合,得到反应混合物,使反应混合物在45℃下转化,而产生红景天苷。结果:在耐热蔗糖磷酸化酶酶液1200 U/L、耐热纤维二糖磷酸化酶酶液500 U/L、蔗糖110 g/L、酪醇30 g/L和磷酸50 m M的浓度下,反应条件为pH 7.0、温度45℃、转速50转/分、反应时间32小时后,红景天苷浓度达到23.7 g/L。结论:本研究使用蔗糖磷酸化酶和纤维二糖磷酸化酶联合催化的工艺,成功地高收率合成了红景天苷。同时,本研究构建的耐热磷酸化酶酶活高,处理简单,为拓展糖苷类似物的合成提供了一种新的方法。

Cloning and Sequence Analysis of rbcS Gene of Wild Barley (Hordeum brevisubulatum) under Salt Stress

[Objective] The aim was to study the cloning and sequence analysis of rbcS gene of wild barley under salt stress. [Method] The tender leaf blade of wild barley under salt stress was taken as the experimental material. The primers were designed according to the homology of rbcS gene sequences of wheat and barely in Genbank; then PCR amplification,recovery,ligation,transformation and sequencing of rbcS gene were carried out. [Result] Two rbcS genes including rbcS1 and rbcS2 with the length of 1 252 and 908 bp respectively were cloned from the barely genome. rbcS1 and rbcS2 were both composed by two exons and one intron. The exons length of the two genes was the same of 525 bp,encoding 174 amino acids,and the homology between them was 96%; however,the intron length of rbcS1 and rbcS2 was 448 and 107 bp respectively.

NUCLEOTIDE SEQUENCE OF RIBULOSE 1,5 BISPHOSPHATE CARBOXYLASE/OXYGENASE LARGE SUBUNIT GENE FROM MILLET(SETARIA ITALICA) *

The 3.0 kb BglⅡ+XbaⅠrestriction fragment of millet(Setaria italica) chloroplast genome containing rbcL gene had been cloned into pBluescript SK (-) vector, then the restriction map and the 1990 bp complete nucleotide sequence was determined. The 1431 bp coding region of the gene consists of 476 amino acid residues with a predicted molecular weight of 52679 D. The 389 bp 5′ upstream region has the putative -10 box, -35 box and SD sequence, similar to that of procaryotes. The 170 bp 3′ downstream region contains three stem loop structures. Comparison of the rbcL gene sequences between C 4 plants and several C 3 plants reveals no difference in the coding region, promoter and 3′ downstream region. It might be concluded that the rbcL gene sequence has no relation with its cell specific expression.

Subcloning and Sequencing of the Form II Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from Rhodopseudomonas palustris

Rhodopseudomonas palustris, one of purple nonsulfur photosynthetic bacteria, fixes carbon dioxide via Calvin-Benson cycle and has been shown previously to express form I and form II ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). The gene cbbM, which encodes the form II enzyme from Rhodopseudomonas palustris, has been subcloned and sequenced. The deduced amino acid sequence is highly with the form II RubisCO from photosynthetic bacteria, including Rhodospirillum rubrum (PDB ID: 9rub), but appears to be more distantly related to the large subunit of the form I RubisCO found in photosynthetic bacteria, chemoautotrophic bacteria and higher plants. Several regions highly conserved among L 8S 8 and L x enzymes correspond with regions previously implicated in catalytic activity and subunit interactions.

Coronatine Induces an Accumulation of Anthocyanin and Starch in Purple-fleshed Sweetpotato(Ipomoea batatas Lam.)

[Objective] The objective of this research was to examine the effects of COR on anthocyanin and starch content in storage roots of two PFS genotypes, and to explore the relationships between anthocyanin synthesis and starch accumula- tion. [Method] A field experiment was carried out to determine the changes in yielc components, yield, contents of anthocyanin and starch, activities of phenylalanine ammonia-lyase （PAL） and adenosine 5-diphosphate glucose pyrophosphorylase （AG- Pase） in two genotypes of PFS （Ipomoea batatas L., var. ＇Ayamurasaki＇ and ＇Jishu18＇）. [Result] The application of COR significantly increased starch and antho- cyanin content in storage roots of Jishu18 across developmental stages by inducing the activities of PAL and AGPase, and finally enhanced yield by promoting fresh weight of storage roots. Ayamurasaki was insensitive to treatment with COR al- though its PAL activity temporally increased. The starch and anthocyanin content of Aya, and the anthocyanin content of Jishu18 increased progressively across devel- opmental stages with or without COR application, but the starch content of Jishu18 increased initially, then decreased before increasing again without application of COR. Treatment with COR reduced downward trend of starch accumulation in Jishu18. Thus, the effect of COR on accumulation of anthocyanin and starch in storage roots of PFS differs according to genotypes. [Conclusion] The application of 0.05 μmol/L COR may increase starch and anthocyanin content in PFS genotypes with lower starch and anthocyanin content in storage roots.