摘要
菊粉酶专一性水解β-2,1-D-呋喃果糖苷键,按其作用方式不同分为内切和外切两类。内切菊粉酶(endoinulinase, EC3.2.1.7)从菊粉链内部随机地切断糖苷键,降解产物主要为菊粉型低聚果糖。外切菊粉酶(exoinulinase, EC3.2.1.80)则从菊粉链的果糖末端逐个地水解糖苷键,产物主要为果糖。菊粉酶广泛存在于各种微生物中,在饲料、食品、医药以及能源工业中有着广阔的应用前景。
本实验室从土壤中成功筛选到一株产菊粉酶的菌株Aspergillus ficuum JNSP5-06,此菌株可以产内切菊粉酶(endo I)和外切菊粉酶(exo I),且产酶性能稳定。为了进一步提高酶活力,探讨菊粉酶作用机制,本研究运用分子生物学的方法,克隆和表达了内、外切菊粉酶的基因,并在此基础上,通过定向诱变技术研究了内切菊粉酶的催化活性中心和菊粉酶分子的C端结构域的作用。
根据Genbank中记录的菊粉酶基因序列信息,利用PCR技术从Aspergillus ficuum JNSP5-06基因组中扩增和克隆了相关基因片段,获得了endo I的DNA和exo I的DNA、cDNA序列,克隆的endo I和exo I的DNA序列在Genbank中的登录号分别为FJ984582和HM587130。序列分析可知:内切菊粉酶endo I的DNA序列全长1658bp,包含了蛋白质编码区1482bp,3′非编码区176bp;内切酶由493个氨基酸组成,理论分子量Mw为53.2kDa,等电点pI为4.33。外切菊粉酶exo I的DNA序列全长1674bp,包含信号肽编码区57bp,蛋白质编码区1557bp,内含子编码区60bp;exoI全长537个氨基酸,其中信号肽和成熟肽分别由19和518个氨基酸残基组成;其理论分子量Mw为57.2kDa,等电点pI为4.89。
将内、外切菊粉酶成熟肽的编码序列分别插入到大肠杆菌表达载体pET-28a(+)中,构建重组质粒pET28a(+)-endo I和pET28a(+)-exo I,转化大肠杆菌BL21(DE3),筛选获得基因工程重组菌JPE21/endo I和JPE21/exo I。对菌株的IPTG诱导表达条件优化结果发现:JPE21/endo I在OD600为0.308左右时,加入IPTG至终浓度0.4mmol/L,23℃诱导6h,酶活最高达22.36u/mg;JPE21/exo I则在OD600为0.402左右时,加入IPTG至终浓度为0.4mmol/L,26℃诱导7h,其酶活可达59.24u/mg。经SDS-PAGE检测,重组内、外切菊粉酶的分子量分别约为59kDa、63kDa。对其酶学性质的研究结果表明:(1)重组内切菊粉酶的最适pH为5.0,最适反应温度为55℃。Ag+和Cu~(2+)能完全抑制酶的活性,Fe~(2+)、Fe3+和Al3+对酶有较强的抑制作用,而Zn~(2+)、Mn~(2+)、K+对酶有激活作用。在pH5.0、55℃条件下,内切酶水解菊粉的Km和kcat分别为(8.9±1.5)mM和(1.38±0.12)×103min-1。(2)重组外切菊粉酶作用于菊粉和蔗糖的最适pH值分别为4.0和5.0;最适温度分别为60℃和55℃。金属离子对其活力的影响与底物相关,Cu~(2+)完全抑制外切酶对菊粉的作用,而对蔗糖保持10%的活性;Ag+使酶对菊粉和蔗糖的活力分别降低了45.6%和82.9%;Mg~(2+)、Zn~(2+)、Fe~(2+)、Al3+、Ni~(2+)对酶有较强的抑制作用,而Mn~(2+)对酶有激活作用。在pH5.0、55℃条件下,外切酶水解菊粉Km和kcat分别为(7.1±0.2)mM和(6.1±0.0)×104min-1,水解蔗糖的Km和kcat分别为(347.6±25.9)mM和(7.34±0.49)×105min-1。
通过酶活测定以及酶解产物的薄层层析和液相色谱分析,发现重组内切菊粉酶仅专一性水解菊粉,产物主要为低聚二糖、三糖、四糖;外切菊粉酶不仅能水解菊粉,还能降解蔗糖,另外在高浓度的蔗糖中,还有微弱的转果糖基作用。
对内切菊粉酶endo I进行同源建模和序列比较,定点突变证明内切菊粉酶催化位点的关键氨基酸残基为Glu-20及Glu-210,底物结合位点的关键氨基酸残基为Asp-153。
利用PCR和overlap-PCR技术使得内、外切菊粉酶的C端结构域发生缺失和交换突变,结果发现突变酶的活性急剧下降,且内切突变酶对蔗糖产生降解作用,推测C端结构域起到识别底物、稳定酶分子空间结构、保持酶催化活性的作用。
Inulinases, which can be divided into exo-and endoinulinase according to their hydrolysis patterns,hydrolyze β-2,1-glycosidic linkages of the fructan. Endoinulinase (2,1-β-D-fructan fructanohydrolase, EC3.2.1.7) acts only on inulin and hydrolyze internal linkages of inulin to yield fructooligosaccharidesrandomly. Exoinulinase (β-D-fructan fructohydrolase, EC3.2.1.80) can hydrolyze terminal linkages ofinulin to yield fructose successively. Iinulinase broadly exists in various microorganisms and has beenwidely used in feed, food, medicine and energy industries.
A strain of Aspergillus ficuum JNSP5-06which could secrete endoinulinase (endo I) and exoinulinase(exo I) was got from the soil in our laboratory. In order to improve the activity of A. ficuum JNSP5-06inulinase and to elucidate the catalytic mechanism of the enzyme, we cloned and expressed the gene. Thenwe studied the catalysis activity centres and the function of C-terminal in the molecular structure ofinulinase through directional mutation technique.
According to the information of inulinase in Genbank, the DNA and cDNA of endo I and exo I werecloned from Aspergillus ficuum JNSP5-06. And the coding gene sequences of endo I and exo I wereregistered in the GenBank (FJ984582and HM587130). The DNA sequence of endo I is1658bp, whichcontains1482bp of protein coding region and176bp of3’ noncoding region. Endo I consists of493aminoacids. Mw and pI are53.2kDa and4.33, respectively. The DNA sequence of exo I is1674bp, whichconsists of57bp signal peptide coding region,1557bp of protein coding region and60bp of intron codingregion. Mw and pI are57.2kDa and4.89, respectively.
The encoding sequences of endo I and exo I were inserted into pET-28a(+) respectively. Therecombinant pET28a(+)-endo I and pET28a(+)-exo I were introduced into host E. coli BL21(DE3). Afterinduced by IPTG, the recombinant JPE21/endo I and JPE21/exo I were successfully expressed. The optimalconditions for expression of JPE21/endo I were IPTG concentration of0.4mM, temperature of23°C andreaction time of6h. And the optimal conditions for expression of JPE21/exo I were IPTG concentration of0.4mM, temperature of26°C and reaction time of7h. After optimization, the specific activities ofJPE21/endo I and JPE21/exo I could reach to22.36u/mg and59.24u/mg, respectively.
The recombinant protein was checked by SDS-PAGE, and the Mw of endo I and exo I were59kDaand61kDa, respectively. The optimum pH and temperature of endo were5and60℃. Ag+and Cu~(2+)couldinhibit the activity of endo I thoroughly, and Fe~(2+)、Fe3+and Al3+cound inhibit activity intensively whileZn~(2+)、Mn~(2+)and K+could activate endo I. Kmand kcatof endo I with inulin as substrate were (8.9±1.5) mMand(1.38±0.12)×103min-1respectively. Studies on characterization exo I showed optimum pH andtemperature were4and60℃for inulin, whereas the optimum pH and temperature for sucrose were5and55℃. The influence of the metal ions on the exo I activity was related to the substrate. Cu~(2+)causeddecrease in exo I activity against inulin and sucrose by100%and90%, respectively, while Ag+causeddecrease in exo I activity against inulin and sucrose by45.6%and82.9%. Mg~(2+)、Zn~(2+)、Fe~(2+)、Al3+and Ni~(2+)could inhibit activity intensively while Mn~(2+)could activate the activity. Kmand kcatwere (7.1±0.2) mM and(6.1±0.0)×104min-1for inulin, whereas Kmand kcatfor sucrose were (347.6±25.9) mM and (7.34±0.49)×105 min-1.
Analysis of enzymatic hydrolysis products by TLC and liquid chromatography showed that endo Iuniquely hydrolyzed inulin, which produced disaccharide, trisaccharide and tetrose. While exo I not onlyhydrolyzed inulin, but also degraded sucrose. In addition, exo I had the weak ability of transfructosylation.
Through homology modeling, sequence comparing and site-directed mutagenesis of endo I, it wasproved that Glu-20and Glu-210were catalytically important residues, and Asp-153played an importantrole for substrate recognition in the catalysis reaction.
C-terminal domain was processed by deleted and swapped to orthomutation through over-lap PCR orPCR. It was found that enzyme activities were decreased sharply by C-terminal domain deletion andswapping mutants, and in this case endo I had the ability to degraded sucrose. The results showed thatC-terminal domain had the abililty to recognize long-chain inulin, stable enzyme molecule space structureand keep the enzyme catalysis activity.
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