6-Phospho-{beta}-galactosidases of Gram-positive and 6-phospho-{beta}-glucosidase B of Gram-negative bacteria: comparison of structure and function by kinetic and immunological methods and mutagenesis of the lacG gene of staphylococcus aureus

The 6-phospho-ß-galactosidase of Staphylococcus aureus,Lactococcus lactis and Lactobacillus casei and 6-phospho-ßglucosidaseB of Escherichia coli build a subfamily inside a greater enzymefamily, named the glycosal hydrolase family 1, which, hi addition,contains nine ß-glycosidases of different origins.Kinetic and immunological evidence is provided in this reportwhich strengthens the relationship of the four 6-phospho-ß-glycosidases.It is shown that the 6-phospho-ß-galactosidases and6-phospho-ß-glucosidase B are able to split aromaticß-galactoside phosphates and ß-glucosidephosphates. The turnover numbers of hydrolysis of substrateswith different epimerization at C-4 of the glycon vary up to15-fold only. Two polydonal antisera, one derived against thenative 6-phospho-ß-galactosidase from S.aureus andthe other derived against the 6-phospho-ß-glucosidaseB, cross-reacted with both enzymes. Peptides of the proteinswere separated by reverse phase HPLC. The cross-reacting peptideswere sequenced and shown to be localized at almost the sameposition in the aligned primary structures of both enzymes.An insertion of nine amino adds near these antigenic domainsis unique for the 6-phospho-ß-glycosidases and missingwithin the sequences of the ß-glycoside-specific membersof the family. The lacG gene of a 6-phospho-ß-galactosidasenegative S.aureus mutant was doned into E.coli and sequenced.In the totally inactive mutant protein only the glycine at position332 was changed to an arginine. This amino acid is part of thesequence insertion near the antigenic domain reacting with bothantisera. These data support the assumption that the regionis of great importance for the function of the enzymes and thatit is possible it determines the specificity of the phosphorylatedform of the substrates. In addition, the 6-phospho-ß-galactosidaseof S.aureus was modified by sitedirected mutagenesis of thecorresponding lacG gene hi order to replace residues Glul60and Glu375, which were suspected of being involved hi the generalacid catalysis of substrate hydrolysis, with glutamine residues.The mutant protein 160EQ retained some catalytic activity whilethe protein 375EQ was totally inactive. Glu375 is the activesite nudeophile of the 6-phospho-ß-galactosidase ofS.aureus. It is located in the sequence motif ENG where Glu358was identified as the catalytkally active nudeophile hi theß-glucosidase of Agrobacterium.     

复合诱变法选育生产乳果糖高产菌株的研究

选育生产乳果糖的高产菌株,为生物法（酶法）合成乳果糖提供技术保障。以乳酸克鲁维酵母变种（JN-187）为出发菌,经紫外线诱变选育出水解活性最高的JU74—09突变菌株;然后以JU74—09菌株为出发菌,经亚硝基胍（NTG）诱变选育出转糖基活性最高的JN91—08突变菌株;该菌株经7次传代实验,显示出良好的遗传稳定性。说明紫外线诱变与亚硝基胍（NTG）复合诱变是一种行之有效的用于生产乳果糖菌株选育的诱变方法。     

琥珀酸产生菌的诱变与选育

对琥珀酸产生菌S-1进行紫外线亚硝基胍的复合诱变后,筛选出琥珀酸产量高,遗传性状稳定的菌株S-57,并对其进行激光诱变,筛选出菌株SH-24,相同发酵条件下琥珀酸产量达到21.25g/L,相对于未诱变菌株产量提高了343倍。     

透明质酸产生菌的复合诱变及摇瓶发酵条件的优化

采用紫外线、亚硝酸钠、亚硝基胍复合诱变的方法,得到遗传稳定性好、分子量高、透明质酸产量增幅3倍以上的突变株.在单因素试验的基础上,确定了影响兽疫链球菌透明质酸产量的关键因素为发酵时间、发酵温度和葡萄糖浓度.采用响应面法优化摇瓶发酵的条件,建立影响因子与其响应值之间的关系,结果表明:在摇瓶发酵温度32.47℃、发酵时间22.52h、葡萄糖浓度在65.46g/L条件下,透明质酸产量为0.562mg/mL.     

复合诱变选育表面活性素(surfactin)高产菌株

为筛选抗菌脂肽表面活性素高产菌株,以枯草芽孢杆菌Bacillus subtilis fmbJ224为出发菌株,先后采用氯化锂(LiCl)、亚硝基胍(NTG)、甲基磺酸乙酯(EMS)和链霉素(streptomycin)进行复合诱变。结果表明：经初筛、复筛选育得到1株高产抗菌脂肽菌株LN2-3,其Surfactin产量达到239.2mg/L,比出发菌株Bacillus subtilis fmbJ224(5.25mg/L)提高了44.56倍,且该菌株具有良好的遗传稳定性。提示采用LiCl-NTG复合诱变能较大幅度的提高诱变率,可以获得抗菌脂肽高产菌株。     

高产二十二碳六烯酸隐甲藻的选育及其发酵条件研究

利用亚硝基胍对隐甲藻ATCC30556进行诱变处理,通过30℃高温培养初筛及摇瓶发酵复筛,得到二十二碳六烯酸(DHA)产量稳定提高的突变株LS1057,其DHA占总脂肪酸的含量从出发株的25.78%提高到32.02%。研究摇瓶发酵条件对突变株发酵的影响,结果表明：在初始pH6.5、温度26℃、转速150r/min的发酵条件下培养216h后,其生物量、总油脂产量及DHA产量分别达到14.06、3.14g/L和1.057g/L,分别比出发藻株提高了21.31%、69.73%和121.59%。     

复合诱变选育高效生物破乳剂产生菌及其特性

为提高破乳性能,以一株生物破乳剂产生菌Bacillus mojavensis XH1为出发菌株,进行紫外和亚硝基胍复合诱变处理,经过筛选分离和连续传代得到一株遗传稳定性较好的高效生物破乳剂产生菌突变株XN5．在碳源为10 g／L葡萄糖和4％ (体积分数)液体石蜡的混合碳源、氮源为40 g／L NH₄Cl与10 g／L酵母膏的混合氮源、温度30 ℃、初始pH=65、摇床转数为140 r／min的培养条件下,培养24 h后,突变菌株的12 h和24 h破乳率分别为9417％和9867％,比原始菌株XH1提高了6481％和1512％．     

富产琥珀酸米曲霉菌株的诱变选育

以米曲霉AS3.042为出发菌株,经过紫外线诱变后选育出一支琥珀酸产量高的菌株AS-4。以AS-4为出发菌株,采用亚硝基胍诱变后得到一支富产琥珀酸米曲霉诱变菌株ASC-5。该菌株经摇床培养后,琥珀酸产量达到3.60 g/L,相同条件下为出发菌株的371倍,为单用紫外线诱变后的菌株AS-4的13.43倍。     

紫外线与亚硝基胍复合诱变选育高效降解亚硝酸盐乳酸菌

目的 通过诱变方法获得高效降解亚硝酸盐的优良乳酸菌应用于降低腌制品中的亚硝酸盐。方法 以前期实验筛选获得的降解亚硝酸盐性能较强乳酸菌D2作为初始诱变菌株, 采用紫外线和亚硝基胍复合诱变, 选育高效降解亚硝酸盐的乳酸菌。结果 经15 W紫外线和0.5 mg/mL亚硝基胍三轮复合诱变得到一株优良乳酸菌, 该菌株24 h降解亚硝酸盐(200 mg/L)降解率为91.4%, 较初始菌株提高了12.7%; 以亚硝酸钠为底物, 其产亚硝酸盐还原酶的比活力为7.7 mmol/L, 较诱变前提高42.9%; 连续传代培养后降解亚硝酸盐能力和产亚硝酸盐还原酶活力性能稳定。结论 通过紫外线和亚硝基胍复合诱变, 获得一株遗传稳定性良好的高效降解亚硝酸盐的菌株。     

富硒酵母的选育及其培养基组分优化研究

硒是人和动物体的一种必需微量元素。有机硒具有吸收快和毒性小等优点,已成为食品和饲料的首选添加剂。酵母菌具有较强的富集硒并将无机硒转化为有机硒的能力。文章以生产性状已退化的酵母菌为出发菌株,通过亚硝基胍诱变选育,筛选出了一株高产稳定的菌株nw-13,并用正交试验优化了培养基组分,最后对其遗传稳定性进行了验证。结果表明,nw-13的生物量为15.8 g/L,有机硒含量为1754μg/g,均高于同类报道;最适培养基组分为:蔗糖6%,牛肉膏1%,蛋白胨1%,K2HPO40.15%,硒浓度40μg/mL;其遗传稳定性较高。为工业生产提供优良的菌种。