Recent advances in Levansucrase and Inulosucrase: evolution,characteristics, and application

Abstract

Levan and inulin are two types of fructan. Levan is composed of β-(2, 6) fructosyl linkage and inulin is composed of β-(2, 1) linkage. Both levan and inulin have been accepted and applied in the food, medicinal and chemical industries for their outstanding physicochemical properties in recent years. Microbial levansucrase and inulosucrase are key enzymes responsible for the synthesis of fructan from sucrose. In this review, levansucrase and inulosucrase are discussed together for the first time regarding the evolutionary relationships, bacteria origin, crystal structure, product-forming mechanism and commercial applications. Particularly, some insights into the product specificity about levansucrase and inulosucrase as well as the mechanism for product elongation for fructan are also discussed in the article.     

The targeting of bacillus subtilis levansucrase in yeast is correlated to both the hydrophobicity of the signal peptide and the net charge of the N-terminus mature part

We compared the ability of signal sequences from various Bacillus or yeast secreted proteins to direct Bacillus subtilis levansucrase into the secretion pathway of the yeast Saccharomyces cerevisiae. The efficiency of these sequences correlated with the overall hydrophobicity of their h-domain and was independent of their origin. Furthermore, the net charge of the proximal protein sequence downstream from the signal sequence contributed to the competence of the heterologous proteins to be secreted by yeast. Modification of this net charge allowed the protein to be translocated under the control of the yeast invertase signal sequence. Moreover, glycosylation of levansucrase did not modify significantly the fructosyl polymerase activity.     

RT-qPCR分析拷贝数及mRNA转录水平对表达左聚糖蔗糖酶的影响

采用实时荧光定量聚合酶链式反应（RT-qPCR）技术对重组毕赤酵母基因组中左聚糖蔗糖酶基因（SacB）的拷贝数及信使核糖核酸（mRNA）转录水平进行检测分析,并用3,5-二硝基水杨酸（DNS）法测定菌株不同诱导时间下左聚糖蔗糖酶水解活力。结果表明,在BMMY液体培养基中经甲醇诱导24 h时,样品转录水平皆达到最大值,此时多拷贝菌株转录水平（2.13）为单拷贝菌株（0.42）的5.1倍;左聚糖蔗糖酶活力在甲醇诱导24 h后均随诱导时间不断上升,多拷贝菌株酶活（13.96 U/mL）较单拷贝菌株（5.48 U/mL）提高1.5倍。重组毕赤酵母整合的左聚糖蔗糖酶基因在1～3个拷贝范围内,随着拷贝数增加,多拷贝菌株较单拷贝菌株的mRNA转录水平及相应的蛋白表达量均显著增加（P＜0.05）,说明毕赤酵母是表达基因SacB的良好宿主。     

Extracellular levansucrase of Bacillus subtilis produced in yeast remains in the cell in its precursor form

Levansucrase, a Bacillus subtilis extracellular enzyme, was not secreted in the culture medium when produced in yeast. The protein accumulated inside the cell in its precursor form which represented 0·3% of total proteins. The absence of any post-translational modifications, such as signal sequence cleavage or addition of N-linked sugars, indicated that this protein did not enter the reticulum secretion pathway. Direct observation of the cells by confocal laser scanning microscopy showed that levansucrase was associated with the cytoplasmic membrane. Subcellular fractionation experiments revealed that levansucrase precursor form is associated with membranes through weak ionic interactions. The purified precursor displayed the same catalytic properties as levansucrase secreted by B. subtilis. Thus yeast could be used as a source of levansucrase precursor allowing its isolation as a pure form on a milligram scale.     

具有极高热稳定性的β-（2，6）果聚糖蔗糖酶及其应用

挖掘具有高热稳定性的β-（2，6）果聚糖蔗糖酶（levansucrase，LS），并应用果聚糖蔗糖酶进行β-（2，6）果聚糖的高效合成。以分子动力学模拟的方式筛选出具有潜在高热稳定性的β-（2，6）果聚糖蔗糖酶。将目的酶在大肠杆菌Escherichia coli BL21（DE3）中进行重组及诱导表达，并通过镍柱亲和层析进行纯化。在不同温度下进行保温实验以验证LS的热稳定性，并通过条件优化获得产物的高转化率。Cedi-LS在65 ℃时显示出最高活性，远高于已鉴定的其他LS。同时，在45 ℃下保温72 h，重组酶Cedi-LS能够保留90%以上的相对活性；在55 ℃下保温60 h，其保留活性仍可达到初始活性的60%以上，表现出优异的热稳定性。在反应过程中，Cedi-LS可同时生成低聚糖、低相对分子质量的β-（2，6）果聚糖和高相对分子质量的β-（2，6）果聚糖。当温度从65 ℃降至35 ℃时，Cedi-LS倾向于产生HMW-levan，其相对分子质量可达8.4×10⁶。在pH 5.5和35 ℃的条件下，以质量分数30%的蔗糖为底物，加酶量定20 μg/mL，蔗糖转化为β-（2，6）果聚糖的平衡转化率为41.4%。作者鉴定了一种具有高热稳定性的LS，并且这种LS能够高效生产一系列具有不同相对分子质量的果聚糖。