Cloning,expression and characterization of L-cysteine desulfhydrase gene from Pseudomonas sp.TS1138

L-cysteine desulfhydrase(CD)plays an impor-tant role in L-cysteine decomposition.To identify the CD gene in Pseudomonas sp.TS1138 and investigate its effect on the L-cysteine biosynthetic pathway,the CD gene was cloned from Pseudomonas sp.TS1138 by polymerase chain reaction(PCR)method.The nucleotide sequence of CD gene was determined to be 1,215 bp,and its homology with other sequences encoding CD was analyzed.Then the CD gene was subcloned into pET-21a(+)vector and expressed in Escherichia coli(E.coli)by isopropyl-β-D-thiogalacto-pyranoside(IPTG)inducement.The recombinant CD was purified by Ni-NTA His-Bind resin,and its activity was identified by the CD activity staining.The enzymatic proper-ties of the recombinant CD were characterized and its critical role involved in the L-cysteine biosynthetic pathway was also discussed.

原生质体紫外诱变选育L-半胱氨酸高活力转化菌株

对Pseudomonas sp．HJ14的原生质体制备和再生条件进行了研究，并对其原生质体紫外辐照诱变选育L-半胱氨酸高活力转化菌株。在37℃、2mg／mL溶菌酶作用下酶解60min，其原生质体的形成率和再生率分别为78．6％和28．6％。经过紫外辐照诱变Pseudomona sp．HJ-14原生质体，在含DL-2-氨基-△^2-噻唑啉-4-羧酸（DL-ATC）再生平板上筛选抗性菌株，获得1株酶活较高的正突变株B-3，该菌株具有良好的遗传稳定性和酶活稳定性。采用5L罐进行产酶培养，并在pH8．0、DL-ATC·3H2O浓度为10g／L、42℃酶促反应9h，L-半胱氨酸最高产量达4．63g／L，摩尔转化率为76．5％，较HJ-14提高了117．7％。