高效絮凝素毕赤酵母表面展示系统的构建

为了获得高效的脂肪酶毕赤酵母表面展示系统,利用来自酿酒酵母絮凝素蛋白Flo1的N端874个氨基酸残基(FS)和C端的1101个氨基酸残基(FL)作为锚定蛋白分别构建了2套载体系统.带有前肽的米黑根毛霉脂肪酶(ProRML)克隆到构建的2套展示载体中,使米黑根毛霉脂肪酶(RML)分别以N端锚定或C端锚定的方式实现在毕赤酵母细胞表面的展示.利用RMLC端的Flag标签,通过流式细胞术和激光扫描共聚焦显微镜检测2套系统中RML在酵母表面的展示情况.研究发现,N端锚定于酵母表面的展示酶FSR以pNPC为底物时,水解活力达到了105.3U/g,大约为C端锚定的展示酶FLR活力的2倍.同时FSR比FLR具有更宽的温度、pH作用范围和更好的热稳定性.与游离酶和固定化酶相比,展示酶FSR也表现出更为优良的热稳定性.结果提示,基于Flo1N端锚定的展示系统更适合展示活性中心近C端的脂肪酶,推动了展示酶的进一步研究和开发.     

糖基工程酵母表面呈现抗体库的构建及抗真菌β甘露糖型抗体的筛选

目的:通过构建糖基工程酵母表面呈现免疫抗体库,筛选与毕赤酵母表达的禽流感血凝素HA7结合的特异性抗体。方法:利用糖基工程毕赤酵母细胞膜锚定蛋白Sed将人Ig G的Fc段锚定在酵母表面,然后用毕赤酵母表达的HA7免疫小鼠,从小鼠脾脏细胞中扩增抗体轻重链可变区基因构建抗体转化锚定了Fc的糖基工程酵母,构建表面展示免疫抗体库,通过免疫磁珠法筛选与毕赤酵母表达的HA7特异性结合的抗体表达酵母,分析筛选该抗体的特异性结合靶点。结果:构建了多态性良好的抗体表达质粒库,转化糖基工程酵母后筛选得到特异性结合HA7的抗体呈现酵母,Western印迹分析发现该抗体只与糖基化的HA7结合而不与切除糖链后的HA7结合,说明抗体结合区域在HA7的糖链上,进一步实验发现该抗体不与α甘露糖型和哺乳动物复杂型糖型结合,通过β甘露糖苷酶切研究发现抗体结合靶点为酵母表达HA7糖链上的β甘露糖。结论:利用糖基工程酵母构建抗体库,筛选得获得了抗真菌β甘露糖的抗体,动物实验发现抗体具有中和活性。     

蝙蝠SARS样冠状病毒WIV1利用小鼠ACE2受体研究

严重急性呼吸综合征冠状病毒(SARS-CoV)是一种感染人类的高致病性病毒,由蝙蝠SARS样冠状病毒(SL-CoV)演化而来。血管紧张素转换酶2(ACE2)是SARS-CoV受体,影响病毒宿主范围、致病性和种间传播。先前研究表明,SARS-CoV和一些SL-CoV株(如WIV1)可以有效利用人、果子狸、蝙蝠ACE2入侵细胞,而SARS-CoV可以低效利用小鼠ACE2。啮齿动物种类多,分布广,包括多种重要的试验动物模型,不过SL-CoV利用啮齿动物ACE2的研究较少。本研究通过假病毒感染试验,比较了SARS-CoV BJ01株和SL-CoV WIV1株利用人类、果子狸、蝙蝠、小鼠ACE2及其突变体进入细胞的效率,并利用蛋白结合试验比较了BJ01和WIV1受体结合结构域(RBD)结合不同ACE2及其突变体的能力。结果显示,SL-CoV WIV1可以有效利用小鼠ACE2进入细胞,且WIV1 RBD与小鼠ACE2结合效率与人和果子狸ACE2相同,强于蝙蝠ACE2;而不同物种ACE2的L440P突变能显著降低其与BJ01及WIV1的RBD结合的能力,抑制假病毒入侵。研究结果表明,小鼠ACE2是SL-CoV WIV1的功能受体,且ACE2的L440是影响病毒入侵的关键氨基酸位点。本研究有助于进一步了解SL-CoV的受体识别、跨种传播机制,对今后类似冠状病毒的防控具有重要意义。     

抗HER2人源化单克隆抗体在毕赤酵母中的表达及其产物分析

目的:研究抗人表皮生长因子受体2(HER2)人源化单克隆抗体能否在毕赤酵母中表达,并对表达产物进行结构分析和活性测定.方法:合成抗HER2人源化单克隆抗体的全基因,构建轻重链双表达盒表达载体,转入毕赤酵母中;通过表型筛选和诱导表达实验得到抗体表达工程菌,对表达产物进行分离纯化和活性测定.结果:表达产物存在于表达上清中,摇瓶表达水平达到53.7±2.9 mg/L;毕赤酵母表达的抗体的轻重链能够自发通过二硫键装配成正确的抗体结构,其中重链发生了N-糖基化;酵母表达的抗HER2人源化单克隆抗体可以抑制高表达p185(abR2)肿瘤细胞SKBR-3的生长,半数有效浓度为0.17 mg/L.结论:实现了抗HER2人源化单克隆抗体在毕赤酵母中的表达,为毕赤酵母表达系统成为抗体等人用剂量较大的复杂型糖蛋白的大规模、低成本制备平台提供了基础.     

基于处方挖掘与分子动力学模拟筛选严重急性呼吸综合征冠状病毒2潜在抑制剂分子的研究

目的 从中药筛选具有潜在抑制严重急性呼吸综合征冠状病毒2 （SARS-CoV-2） 活性的成分，进一步从原子水平揭 示其抑制SARS-CoV-2 表面刺突蛋白（S 蛋白） 受体结合域（RBD） 与血管紧张素转化酶2 （ACE2） 结合的内在机制。 方法 检索新型冠状病毒（简称“新冠肺炎”） 治疗中药处方，构建“新冠肺炎中药候选活性成分数据库”。用具有ACE2 抑制活性的小分子化合物构建HipHop药效团模型，并对“新冠肺炎中药候选活性成分数据库”中活性成分筛选。采用分子 对接和分子动力学模拟方法研究候选活性成分与ACE2 的结合方式及其对SARS-CoV-2 S 蛋白与ACE2 识别的影响。 结果 本文通过中药处方挖掘和分子动力学模拟，从143 个新冠肺炎治疗中药处方中筛选出10 种可与SARS-CoV-2 S 蛋白/ 人源ACE2 识别位点结合的中药成分。其中，枇杷叶主要活性成分23-trans-p-coumaryhormentic acid 与ACE2 具有最高的亲和 力，且23-trans-p-coumaryhormentic acid 的结合可有效阻断SARS-CoV-2 S蛋白与宿主细胞ACE2 的结合。结论 本文通过虚 拟筛选发现了SARS-CoV-2 潜在抑制剂分子23-trans-p-coumaryhormentic acid，同时从原子水平预测了其抑制SARS-CoV-2 S 蛋白与ACE2 结合的内在机制，这将为SARS-CoV-2 特异性抗病毒药物的研发提供理论依据。     

猪瘟病毒E2蛋白B/C抗原区基因在毕赤酵母中的表达与鉴定

基于猪瘟病毒主要保护性抗原---E2囊膜糖蛋白有两个相对独立的抗原结构单位---B/C抗原区和A/D抗原区 ,设计引物扩增编码猪瘟病毒E2蛋白B/C抗原区的基因 ,将大小为261bp的PCR产物插入含有强启动子P_AOX1和α-MF信号肽序列的巴斯德毕赤酵母 (Pichiapastoris)表达载体pPICZαC中 ,构建成重组质粒pPICZα-BC ,酶切线性化后电穿孔导入巴斯德毕赤酵母菌X₃₃ 中 ,经Zeocin^TM 筛选得到 3株高拷贝转化子 ,甲醇诱导表达 ,SDS-PAGE和Westernblot及ELISA试验表明 ,酵母培养上清液中含有具有良好反应原性的E2蛋白。为研究亚单位疫苗或诊断抗原打下坚实基础。     

酵母表面展示技术应用的最新进展

酵母表面展示(yeast surface display, YSD)技术是一种将外源靶蛋白基因序列与特定的载体基因序列融合后导入酵母细胞，利用酵母细胞内蛋白转运机制将靶蛋白表达并定位于酵母细胞表面的技术，最常用的是α-凝集素表达系统。酵母细胞具有真核细胞翻译后修饰机制，能够帮助目的蛋白正确折叠，可以用来展示各种真核蛋白，包括抗体、受体、酶和抗原肽等。酵母表面展示技术已成为生物技术和生物医学领域的强大蛋白质工程工具，结合流式细胞分选可用于改善蛋白质性质，包括亲和力、特异性、酶功能和稳定性等。本文从文库构建与筛选、抗体工程、蛋白质工程、酶工程和疫苗开发等方面对酵母表面展示技术应用最新进展进行了综述。     

疏棉状嗜热丝孢菌脂肪酶在毕赤酵母中的表面展示及酶学性质

【目的】构建疏棉状嗜热丝孢菌脂肪酶(Thermomyces lanuginosus lipase,TLL)在毕赤酵母GS115中的细胞表面展示体系,筛选展示成功且酶活力及展示率较高的重组子作为全细胞催化剂,并研究其酶学性质。【方法】克隆TLL基因tll,以酿酒酵母细胞壁蛋白Sed1p为锚定蛋白,构建表面展示载体pPICZαA-TLS。重组载体经SacⅠ线性化后转入毕赤酵母GS115中,经三丁酸甘油酯平板检测及摇甁发酵筛选获得高酶活力的毕赤酵母重组子,采用抗FLAG标签一抗和R-PE荧光素标记的二抗处理细胞后,进行荧光显微镜检测和流式细胞仪分析,并考察全细胞催化剂的最适反应温度和pH、金属离子耐受性等酶学性质。【结果】成功构建TLL毕赤酵母细胞表面展示体系,筛选到1株具有三丁酸甘油酯和橄榄油水解活力的克隆子,经1%的甲醇诱导发酵120 h后,水解橄榄油酶活力达257.8 U/g干细胞。经抗体处理后的重组菌发酵细胞在荧光显微镜下呈现强烈的红色荧光,流式细胞仪分析结果也证实脂肪酶被成功展示在酵母细胞表面,展示率达98.36%。展示的TLL作为全细胞催化剂水解对硝基苯酚丁酸酯(pNPB)的最适温度为30℃,最适pH为8.0,且具备良好的热稳定性和有机溶剂耐受性;K+、Ca2+、Mg2+对其有微弱的激活作用,Mn2+、Ni2+则有微弱的抑制作用,Cu2+的抑制作用较强,而EDTA、SDS、Tween 20对酶活力影响不明显。【结论】首次将TLL脂肪酶成功展示在毕赤酵母细胞表面,获得具有较高水解活力和良好酶学特性的全细胞催化剂,为表面展示TLL脂肪酶的规模化应用奠定了技术基础。     

海藻糖合酶在毕赤酵母表面的展示

海藻糖合酶能够利用麦芽糖一步法转化生产海藻糖,其底物专一性较高,该酶体系生产工艺简单,不受底物麦芽糖浓度的影响,是工业生产海藻糖的首选。为获得具有生产海藻糖合酶能力的毕赤酵母表面展示载体,实验以筛选的Pseudomonas putide P06海藻糖合酶基因为模板,PCR扩增得到海藻糖合酶基因(tres,2064 bp),连接至pPICZαA质粒中,获得重组质粒pPICZαA-tres。以来自酿酒酵母的共价连接细胞壁的Pir系列蛋白的Pir1p成熟肽蛋白作为毕赤酵母表面展示的锚定蛋白,利用PCR技术扩增得到pir1p(847 bp),连接至重组质粒pPICZαA-tres中,获得重组质粒pPICZαA-tres-pir1p。将重组质粒电击转入毕赤酵母GS115中,利用α-factor信号肽将蛋白引导分泌至细胞壁展示于毕赤酵母表面。通过Zeocin抗性筛选,挑选出阳性克隆子并摇瓶发酵。发酵产物经离心、破碎并使用昆布多糖酶水解,洗脱,结果显示,SDS-聚丙烯酰胺凝胶电泳分析可见明显融合蛋白条带,表明海藻糖合酶已成功地锚定在毕赤酵母。将重组毕赤酵母使用pH 7.5的缓冲液清洗并重悬,与底物浓度为30%的麦芽糖在30℃~60℃水浴条件下作用2 h,反应产物利用HPLC检测,能够检测到酶学活性。在优化后的条件pH 7.5,50℃,表面展示海藻糖合酶酶活达到300.65 U/g。40℃~50℃酶活较稳定,保温60 min,残留酶活相对活力达75%以上;最适反应pH值为7.5,并在碱性环境下稳定。     

β2肾上腺素受体基因在毕赤酵母中的表达及活性测定

为提高β2－肾上腺素受体(β2AR)表达量,满足生产需求,使其应用到抗体技术上,利用分子克隆技术将β2－肾上腺素受体基因(β2AR)与绿色荧光蛋白基因(eGFP)克隆到毕赤酵母（Pichia pastoris）表达载体中。表达载体pPICZαDNAsGFP转化至酵母后,β2AR和eGFP基因与酵母基因重组。利用筛选出的阳性重组子诱导表达β2AR和eGFP的融合蛋白,通过125I标记的配体结合实验证明获得了有受体活性的融合蛋白。     

重组猪胰蛋白酶及其R122位点突变体在毕赤酵母中的高效表达及其性质研究

目的:研究R122位点突变重组猪胰蛋白酶,与野生型酶相比较,该位点对重组猪胰蛋白酶(RPT)性质的影响。方法:以毕赤酵母GS115作为表达宿主,对RPT、突变体mRPT(R122H)和 mRPT(R122H/R73G/R130T)进行表达及纯化。并对其性质和稳定进行对比研究。结果:重组胰蛋白酶及其突变体在毕赤酵母中均获得了高效表达。相对于RPT,突变体mRPT(R122H)和 mRPT(R122H/R73G/R130T)在以N-苯甲酰-L-精氨酸乙酯 (BAEE)为底物时,具有更强底物结合力,三者的米氏常数分别为18.8μmol/L、9.0μmol/L和11.0μmol/L;两突变体耐高温耐碱能力增强;在Ca ²⁺存在及去除的条件下,突变体具有更强的抗自降解能力。 结论:可以利用毕赤酵母高效表达重组胰蛋白酶及其突变体。mRPT(R122H)和mRPT(R122H/R73G/R130T) 相对于野生型RPT,对高pH条件和高温的耐受性增强,该稳定性的提高主要归因于R122位点的突变。     

毕赤酵母底盘芳香族氨基酸合成途径改造生产肉桂酸及对香豆酸*

目的:改造毕赤酵母使其异源合成类黄酮生物合成途径的重要中间体肉桂酸、对香豆酸,并优化前体芳香族氨基酸生物合成途径以提高毕赤酵母的生产能力。方法:在毕赤酵母GS115中利用乙醇诱导型人工转录系统表达Rhodotorula glutinis来源的苯丙氨酸解氨酶,并在该重组菌株中分别过表达胞内芳香族氨基酸生物合成途径中的关键酶或其突变体以进行优化。结果:异源表达苯丙氨酸解氨酶可使毕赤酵母将自身产生的L-苯丙氨酸、L-酪氨酸转化为肉桂酸(38.8 mg/L)、对香豆酸(34.2 mg/L),而通过过表达相关酶进行优化,最终肉桂酸和对香豆酸的产量分别达到124.1 mg/L和302.0 mg/L。结论:利用新的异源宿主毕赤酵母成功合成了肉桂酸、对香豆酸,并对胞内的芳香族氨基酸生物合成途径进行了优化,表明毕赤酵母具有生产黄酮类化合物的应用潜力,也为其他芳香族氨基酸衍生物或植物化合物在毕赤酵母中的异源合成奠定了基础。     

基于重组毕赤酵母的fusaruside生物合成

目的:构建产fusaruside的毕赤酵母菌株,解决天然小分子免疫抑制剂fusaruside的来源问题。方法:从禾谷镰刀菌Fusarium graminearum PH-1中扩增获得合成fusaruside的相关基因-3位去饱和酶[Δ3(E)-SD]和10位去饱和酶[Δ10(E)-SD]基因;并通过2A肽策略构建两种基因的共表达载体,转化到毕赤酵母GS115中进行双酶的诱导表达;对诱导后的毕赤酵母菌体进行甲醇和二氯甲烷的处理后,经高效液相色谱质谱联用仪(HPLC-MS)检测其中产物变化。结果:3位去饱和酶和10位去饱和酶在毕赤酵母中成功共表达,SDS-PAGE显示3位去饱和酶分子量约为48kDa,10位去饱和酶分子量约为65kDa; HPLC-MS显示重组酵母可以产生fusaruside。结论:与fusaruside原产菌株镰刀菌相比,该酵母菌的发酵时间更短、产量更高,为fusaruside的进一步开发与应用奠定基础。     

桦褐孔菌膜二肽酶在巴斯德毕赤酵母中的分泌表达

本研究利用巴斯德毕赤酵母Pichia pastoris蛋白表达体系表达了药用担子菌桦褐孔菌的一个二肽酶基因。该二肽酶基因编码区全长1814bp,包含6个内含子,编码465个氨基酸。生物信息学分析发现,二肽酶基因编码的蛋白中不含信号肽序列,但在第55–77位氨基酸之间存在一个跨膜结构。将含跨膜结构和去跨膜结构蛋白的cDNA序列分别克隆到酵母分泌型表达载体pPICZαA上,电转化至巴斯德毕赤酵母X-33中,用1%(V/V)甲醇诱导重组菌株表达目标蛋白,采用SDS-PAGE和Western-blot检测表达蛋白。结果显示,巴斯德毕赤酵母可表达含跨膜结构的完整基因,但目标蛋白不能分泌到胞外,存在于破碎细胞的沉淀中,且没有催化活性;而去跨膜结构的蛋白则可分泌表达到胞外,并具有催化活性。Ni-NTA纯化去跨膜结构的桦褐孔菌二肽酶浓度可达0.12mg/mL,并发现其在pH 7.3、反应温度50℃、反应时间2h的条件下,以Gly-Gly为底物时,其比活为433U/mg。同时检测到其对Ile-Leu、Trp-Trp和Phe-Phe具有较高的水解活性。     

FMRFamide receptors in Helix aspersa

An in vitro receptor binding assay and an isolated heart bioassay were developed and used to characterize the structure-activity relations (SAR) of FMRFamide receptors in a land snail, Helix aspersa. In the radioreceptor assay, binding of ¹²⁵I-desaminoTyr-Phe-norLeu-Arg-Phe-amide (¹²⁵I-daYFnLRFamide) at 0°C to Helix brain membranes was reversible, saturable, and specific, with a K_D of 14 nM and a B_max of 85 fmol/mg brain. A lower affinity site was also observed (K_D=245 nM; B_max=575 fmol/mg brain). In the heart bioassay, daYFnLRFamide and other FMRFamide analogs increased myocardial contraction force. The SAR of cardiostimulation correlated with the specificity of high affinity ¹²⁵I-daYFnLRFamide binding to brain and heart receptors. The SAR was also similar to that described for other molluscan FMRFamide bioassays, except for a marked preference for N-blocked analogs. Peptides with N-terminal extensions of desaminoTyr, Tyr, Tyr-Gly-Gly, and acetyl, exhibited the highest potency in both radioligand displacement and cardiostimulation. The endogenous Helix heptapeptide analogs of FLRFamide (pQDP-, NDP-, and SDP-FLRFamide) were stimulatory on the heart at low doses, but were inhibitory at moderate to high doses. These peptides were 20 times weaker than FMRFamide in both the brain and heart receptor binding assays, with IC₅₀s about 10 μM. The results suggest that the effects of FMRFamide in Helix are receptor-mediated, and that the heptapeptides do not interact at FMRFamide receptors.     

Biophysical Fitness Landscape of the SARS-CoV-2 Delta Variant Receptor Binding Domain

Among the five known SARS-CoV-2 variants of concern, Delta is the most virulent leading to severe symptoms and increased mortality among infected people. Our study seeks to examine how the biophysical parameters of the Delta variant correlate to the clinical observations. Receptor binding domain (RBD) is the first point of contact with the human host cells and is the immunodominant form of the spike protein. Delta variant RBD contains two novel mutations L452R and T478K. We examined the effect of single as well as the double mutations on RBD expression in human Expi293 cells, RBD stability using urea and thermal denaturation, and RBD binding to angiotensin converting enzyme 2 (ACE2) receptor and to neutralizing antibodies using isothermal titration calorimetry. Delta variant RBD showed significantly higher expression compared to the wild-type RBD, and the increased expression is due to L452R mutation. Despite their non-conservative nature, none of the mutations significantly affected RBD structure and stability. All mutants showed similar binding affinity to ACE2 and to Class 1 antibodies (CC12.1 and LY-CoV016) as that of the wild-type. Delta double mutant L452R/T478K showed no binding to Class 2 antibodies (P2B-2F6 and LY-CoV555) and a hundred-fold weaker binding to a Class 3 antibody (REGN10987), and the decreased antibody binding is determined by the L452R mutation. These results indicate that the immune escape from neutralizing antibodies, rather than increased receptor binding, is the main biophysical parameter that determined the fitness landscape of the Delta variant RBD.     

Two-step ion-exchange chromatographic purification of recombinant hirudin-II and its C-terminal-truncated derivatives expressed in Pichia pastoris

The scene of the protein micro-heterogeneity of recombinant hirudin-II (HV2) expressed in Pichia pastoris was investigated. It was shown that three derivatives of HV2 were present in the fermentation broth of P. pastoris, which were intact HV2 and its two derivatives truncated the C-terminal amino acid residue Gln and Leu-Gln, respectively. To purify the minor degradation derivatives of HV2, a simple, biocompatible and scale-up-feasible purification process with two-step ion-exchange chromatography was established instead of usual reverse phase chromatography. The purities of end products were over 96% and the residual endotoxin less than 0.5 EU/ml.     

Production and purification of a novel thermostable phytase by Pichia pastoris FPHY34

A genetically engineered Pichia pastoris FPHY34 strain containing a 1.3 kb thermostable phytase gene (fphy) evolved by DNA shuffling was constructed and screened. Expression and purification conditions for the recombinant phytase were developed in this study. The effect of Pi on recombinant phytase expression and cell growth of P. pastoris FPHY34 was tested in shake flask culture. Optimization of carbon sources for cell growth and methanol feeding strategies for phytase expression in P. pastoris FPHY34 was carried out in a 50-L fermenter by fed-batch fermentation. The purification of phytase was investigated by micro-filtration and ultra-filtration followed by desalting, ion-exchange chromatography, and gel filtration in the ÄKTA system. It showed that the optimum inorganic phosphorus is 13.6 g L⁻¹ and that glucose can be used as a substrate for P. pastoris cell growth instead of glycerol; the biomass yield of glycerol (Y_X/S) is slightly higher than that of glucose. Different profiles of lag phase and respiratory quotient (RQ) displayed between glucose and glycerol as the sole carbon source. The maximum phytase activity in per millimetre reached 2508 U mL⁻¹ at a methanol feed rate of 3.0 mL L⁻¹ h⁻¹ after 80 h period of induction. A purification factor of 41.1 with a 32% yield was achieved after chromatographic purification. The specific enzyme activity was 80 U mg⁻¹ and 3281 U mg⁻¹ in that supernatant fraction and after gel filtration purification, respectively. The strain P. pastoris FPHY34 showed a promising application in phytase industrial production.     

Multimerization- and glycosylation-dependent receptor binding of SARS-CoV-2 spike proteins

Receptor binding studies on sarbecoviruses would benefit from an available toolkit of recombinant spike proteins, or domains thereof, that recapitulate receptor binding properties of native viruses. We hypothesized that trimeric Receptor Binding Domain (RBD) proteins would be suitable candidates to study receptor binding properties of SARS-CoV-1 and -2. Here we created monomeric and trimeric fluorescent RBD proteins, derived from adherent HEK293T, as well as in GnTI-/- mutant cells, to analyze the effect of complex vs high mannose glycosylation on receptor binding. The results demonstrate that trimeric, complex glycosylated proteins are superior in receptor binding compared to monomeric and immaturely glycosylated variants. Although differences in binding to commonly used cell lines were minimal between the different RBD preparations, substantial differences were observed when respiratory tissues of experimental animals were stained. The RBD trimers demonstrated distinct ACE2 expression profiles in bronchiolar ducts and confirmed the higher binding affinity of SARS-CoV-2 over SARS-CoV-1. Our results show that complex glycosylated trimeric RBD proteins are attractive to analyze sarbecovirus receptor binding and explore ACE2 expression profiles in tissues.     

Inhibition of soil-borne plant pathogens by the treatment of sinigrin and myrosinases released from reconstructed Escherichia coli and Pichia pastoris

Myrosinases (thioglucoside glucohydrolase, EC 3.2.3.1) are able to hydrolyse glucosinolates in natural plant products. In Arabidopsis thaliana three different genes with different tissue-specific expressions and distribution patterns encode myrosinases. cDNAs of myrosinase genes (TGG1 and TGG2) were isolated from A. thaliana and expressed in Escherichia coli and Pichia pastoris. The enzyme activities of myrosinase TGG1 and TGG2 genes expressed in P. pastoris were higher than those expressed in E. coli. Among six glucosinolates tested for specificity to myrosinases TGG1 and TGG2, the suitable substrates for these two genes expressed in P. pastoris and E. coli were sinigrin, gluconapin, glucobrassicanapin and glucoraphanin. Treatment of sinigrin with myrosinases excreted from reconstructed E. coli and P. pastoris with TGG1 and TGG2 genes showed strong fungicidal effects on mycelial growth of Rhizoctonia solani AG-4, Sclerotium rolfsii, and Pythium aphanidermatum. This study suggests that the combination of glucosinolate with myrosinases excreted from the reconstructed microbes may be of potential for control of soil-borne diseases.