Improvement on production of (R)-4-chloro-3-hydroxybutyrate and (S)-3-hydroxy-gamma-butyrolactone with recombinant Escherichia coli cells

(R)-4-Chloro-3-hydroxybutyrate (CHB) and (S)-3-hydroxy-gamma-butyrolactone (HL) are used for the synthesis of biologically and pharmacologically important compounds. Enterobacter sp. DS-S-75 was found to have the unique activity to convert (S)-CHB in the racemate to (S)-HL through asymmetric dechlorination, hydrolysis, and lactonization. As a result, the remaining (R)-CHB and formed (S)-HL could be obtained in a one-pot reaction. We purified the CHB degrading enzyme which catalyzing these reactions and isolated the coding gene from the strain DS-S-75 in order to improve the productivity of these compounds using the transformant. Interestingly, the purified enzyme showed not only dechlorinating, but also hydrolyzing activities on CHB and the similar carboxylic esters, it was then designated CHB hydrolase, and appears to be a novel enzyme. The gene had 1101 bp encoding 367 amino acids including a signal peptide composed of 25 residues. The deduced amino acid sequence contained a conserved region generally found in esterases and lipases, but did not have significant similarity. When asymmetric degradation of racemic methyl CHB (CHBM) was performed using a culture broth of Escherichia coli DH5alpha transformed with the isolated gene, the reaction time was shortened 20-fold over that of the strain DS-S-75, and the maximum concentration of the substrate could be increased from 8% to 15% (w/v). Moreover, both of the obtained residual (R)-CHBM and the formed (S)-HL had high optical purities (>99% e.e.).     

Lipase-catalyzed kinetic resolution of 2,3-epoxy-1-tridecanol and its application to facile synthesis of (+)-disparlure

Acylation of (+/-)-2,3-epoxy-1-tridecanol with acetic anhydride in diisopropyl ether by porcine pancreatic lipase yielded (2R, 3S)-2,3-epoxy-1-tridecanol as the remaining substrate with an optical purity of over 99% ee. (+)-Disparlure was synthesized in two steps from this optically active epoxy alcohol.     

A novel lactic acid bacterium for the production of high purity L-lactic acid, Lactobacillus paracasei subsp. paracasei CHB2121

Fermentation-derived lactic acid has several potential industrial uses as an intermediate carbon chemical and a raw material for biodegradable polymer. We therefore undertook the identification of a novel bacterial strain that is capable of producing high concentrations of lactic acid and has potential commercial applications. A novel L(+)-lactic acid producing bacterium, Lactobacillus paracasei subsp. paracasei CHB2121 was isolated from soil obtained near an ethanol production factory and identified by 16S rRNA gene sequence analysis and characterization using an API 50 CHL kit. L. paracasei subsp. paracasei CHB2121 efficiently produced 192 g/L lactic acid from medium containing 200 g/L of glucose, with 3.99 g/(L·h) productivity, and 0.96 g/g yield. In addition, the optical purity of the produced lactic acid was estimated to be 96.6% L(+)-lactic acid. The newly identified L. paracasei subsp. paracasei CHB2121 efficiently produces high concentrations of lactic acid, and may be suitable for use in the industrial production of lactic acid.     

Expression of a gene encoding chitinase (pCA 8 ORF) from Aeromonas sp. no. 10S-24 in Escherichia coli and enzyme characterization

A gene encoding chitinase from Aeromonas sp. no. 10S-24 was expressed using pTrc99A in Escherichia coli JM 105 which yielded a 5-fold higher activity than when pUC19 was used. Three different truncated enzymes (SA-1, SA-2 and SA-3) were obtained after purification. Their isoelectric points were 7.0, 6.9, and 6.7, respectively. The enzymes showed two optimum pHs, 4.0 and 7.0, when incubated with ethylene glycol chitin as the substrate, and were stable over a wide pH range (3.0-9.0). The optimum temperature was 60 degrees C and the enzymes were stable up to 50 degrees C. The chitinases exhibited wide substrate specificities for chitin-related compounds.     

Double knockout of β-lactamase and cephalosporin acetyl esterase genes from Escherichia coli reduces cephalosporin C decomposition

The phenomenon of CPC decomposition occurs in Escherichia coli JM105/pMKC-sCPCacy during the one-step enzymatic conversion of cephalosporin C (CPC) into 7-aminocephalosporanic acid (7-ACA) by CPC acylase (sCPCAcy) for synthesis of cephalosporin antibiotics. E. coli JM105/pMKC-sCPCacy can constitutively produce sCPCacy as a fusion protein with maltose binding protein (MBP). Control experiments verified that the cell lysis solution from the host E. coli JM105 resulted in CPC decomposition by approximately 15%. Two miscellaneous enzymes, β-lactamase (AmpC) and cephalosporin acetyl esterase (Aes), are believed to play a major role in the degradation of CPC. Using the Red recombination system, the genes ampC, aes or both ampC and aes were knocked out from the chromosome of E. coli JM105 to generate the engineers: E. coli JM105(ΔampC), E. coli JM105(Δaes) and E. coli JM105(ΔampC, Δaes). The CPC decomposition was reduced to 12.2% in E. coli JM105(Δaes), 1.3% in E. coli JM105(ΔampC), and even undetectable in ampC-aes double knockout cells of E. coli JM105(ΔampC, Δaes). When catalyzed by crude MBP-sCPCAcy isolated from E. coli JM105(ΔampC, Δaes)/pMKC-sCPCacy (3377U·l(-1)), the CPC utilization efficiency increased to 98.4% from the original 88.7%. Similar results were obtained for the ampC-aes double knockout host derived from E. coli JM109(DE3) and the CPC utilization efficiency enhanced to 99.3% in the catalysis of crude sCPCAcy harvested from E. coli JM109(DE3, ΔampC, Δaes)/pET28-sCPCacy.     

Expression of Rhodococcus opacus alkB genes in anhydrous organic solvents

Rhodococcus opacus B-4 is a benzene-tolerant bacterium which was isolated from a gasoline-contaminated soil sample. We previously demonstrated that this organism was able to survive and exhibit biocatalytic activity in anhydrous organic solvents for at least 5 d. In the present study, we cloned the alkB1 and alkB2 genes encoding alkane hydroxylases from R. opacus B-4. Heterologous expression of the alkB1 and alkB2 genes in Escherichia coli JM109 showed that they encode functional alkane hydroxylases with a substrate range of C(5)-C(16). Promoters of the alkB1 and alkB2 genes, designated P(alkB1) and P(alkB2), respectively, were examined for activity in anhydrous bis (2-ethylhexyl) phthalate (BEHP) containing C(5)-C(16)n-alkanes. Two recombinant plasmids, pP(alkB1)EGFP and pP(alkB2)EGFP, were constructed by inserting the egfp gene downstream of P(alkB1) and P(alkB2), respectively and transformed into R. opacus B-4. Resting cells of R. opacus B-4 (pP(alkB1)EGFP) showed greater levels of EGFP fluorescence in anhydrous BEHP than in 0.85% NaCl, when C(8)-C(16)n-alkanes were supplied as an inducer. Furthermore, n-alkane inducibility of P(alkB1) activity in anhydrous BEHP was noticeably different from that in 0.85% NaCl. This paper presents the first evidence that bacteria can express their genes in essentially anhydrous organic solvents.     

耐热木聚糖酶和葡萄糖醛酸苷酶的共表达及应用

目的：提高海栖热袍菌（Thermotoga maritima MSB8）来源的木聚糖酶XynB和α-葡萄糖醛酸苷酶AguA生产效率并降低生产成本。方法：利用基因重组技术将海栖热袍菌的XynB和AguA基因置于不同表达盒下构建共表达载体pET-20b-xynB-aguA和pET-28a-xynB-aguA,分别转化大肠杆菌Escherichia coli JM109（DE3）进行诱导表达,获得双酶混合物进而水解桦木木聚糖及玉米芯。结果：重组菌E. coli JM109（DE3）/pET-28a-xynB-aguA比E. coliJM109（DE3）/pET-20b-xynB-aguA产酶更具优势,在LB培养基中诱导培养8 h,XynB和AguA的产量分别达到7.6 U/mL和0.5 U/mL,在TB培养基中培养,XynB可达到10.27 U/mL,AguA为1.5 U/mL。在80 ℃水解条件下,双酶比单一木聚糖酶能够更彻底降解桦木木聚糖,所得酶解液中木二糖的含量和纯度更高;从还原糖释放量及电子显微镜观察可以看出双酶液对农副产品玉米芯具有良好的降解作用。结论：XynB和AguA基因（T. maritima）的克隆共表达具有可行性,并且在生物转化、食品工业和饲料生产等领域具有潜在应用前景。     

Expression of a gene encoding chitinase (pCA 8 ORF) from Aeromonas sp. no. 10S-24 in Escherichia coli and enzyme characterization

A gene encoding chitinase from Aeromonas sp. no. 10S-24 was expressed using pTrc99A in Escherichia coli JM 105 which yielded a 5-fold higher activity than when pUC19 was used. Three different truncated enzymes (SA-1, SA-2 and SA-3) were obtained after purification. Their isoelectric points were 7.0, 6.9, and 6.7, respectively. The enzymes showed two optimum pHs, 4.0 and 7.0, when incubated with ethylene glycol chitin as the substrate, and were stable over a wide pH range (3.0–9.0). The optimum temperature was 60°C and the enzymes were stable up to 50°C. The chitinases exhibited wide substrate specificities for chitin-related compounds.     

A novel enzymatic approach to the massproduction of L-galactose from L-sorbose

Wild-type strain of Pseudomonas cichorii ST-24 was unable to grow on D -psicose and inductively produced D -tagatose 3-epimerase (D -TE) with D -tagatose as an inducer. We have isolated a constitutive mutant, designated strain Ka75, which had acquired a new ability to grow on a mineral salts medium containing D -psicose as a sole carbon source. The D -psicose-metabolizing mutant synthesized a high level of D -TE. When grown on the culture medium supplemented with Mn(2+), the mutant strain produced around 250-fold higher activity than did the parent strain. Enzymatic properties of the constitutive enzyme were similar to those of the wild-type. Using the immobilized D -TE and recombinant L-rhamnose isomerase (L-RhI) from Escherichia coli strain JM109, a two-step enzymatic reaction was performed for massproduction of a rare aldo-hexose monosaccharide, L-galactose, from a common one, L-sorbose. In the first step, L-sorbose was epimerized to L-tagatose in a yield of 28%. The L-tagatose obtained was utilized as a starting material for L-galactose preparation by the immobilized L-RhI. At equilibrium, approximately 30% L-tagatose was isomerized to L-galactose. Finally, 7.5 g of L-galactose was obtained from 100 g of L-sorbose, viz an overall yield of 7.5%. The product obtained was purified and identified to be L-galactose by specific optical rotation and high performance liquid chromatography (HPLC) analysis, and was ultimately confirmed by (13)C nuclear magnetic resonance ((13)C NMR) and IR spectra.     

1 株水开菲尔粒中植物乳杆菌的鉴定及产细菌素基因分析

目的：对从水开菲尔粒中分离得到1 株能产生抑菌物质的菌株QF01进行菌种鉴定,并对其产细菌素进行实验、鉴定和基因序列分析。方法：通过牛津杯法测定抑菌能力,采用聚合酶链式反应（polymerase chain reaction,PCR）扩增16S rDNA和细菌素相关基因并测序,利用ProParam tool、TMHMM 2.0、InterProScan、SOPM和SWISS-MODEL在线软件对基因编码产物进行分析。结果：该菌株产的细菌素对大肠杆菌（Escherichia coli JM109）有明显的抑制作用,通过16S rDNA序列分析初步确定该菌株属于植物乳杆菌（Lactobacillus plantarum）。该菌株含有plnD、plnEF、plnV、plnR四种基因,其中plnV基因编码产物有跨膜螺旋结构,其结构存在信号肽特征。此外,从三级结构的模型预测得到4?种基因的编码产物均存在α-螺旋、β-转角、伸展链和无规则卷曲。结论：从水开菲尔粒分离得到的L. plantarum QF01菌株,含有plnD、plnEF、plnV、plnR细菌素基因,对大肠杆菌有很好的抑制作用。     

Asymmetric reduction of ethyl acetoacetate to ethyl (R)-3-hydroxybutyrate coupled with nitrate reduction by Paracoccus denitrificans

We found that the asymmetric reduction of ethyl acetoacetate (EAA) to ethyl (R)-3-hydroxybutyrate (EHB) by Paracoccus denitrificans could be induced by nitrate addition under anaerobic conditions. However, the addition of electron donors such as glucose, ethanol, and methanol together with nitrate did not stimulate EHB production from EAA. When the reaction was carried out under optimum conditions (cell concentration, 10 g-d.w.l(-1); EAA, 150 mM; NO3-, 100 mM), after 8 h of reaction 49 mM of EHB with an optical purity of 98.9% e.e. was produced. Furthermore, a fed-batch reaction was carried out with an intermittent addition of nitrate and EAA to reduce substrate inhibition. The linear reduction of EAA to EHB proceeded for 40 h after initiation of the reaction, and finally 124 mM of EHB with an optical purity of 88.7% e.e. was produced after 104 h.     

酒曲中产凝乳酶微生物菌株的分离筛选及鉴定

针对酒曲中的微生物进行分离纯化,得到11株细菌和2株真菌,并采用酪蛋白平板法和Arima时间法筛选出了1株产凝乳酶的细菌菌株编号为LB-51。通过形态学观察、生理生化实验和16S rDNA序列分析鉴定该菌株为解淀粉芽孢杆菌,将该产凝乳酶菌株命名为解淀粉芽孢杆菌GSBa-1。该菌株在液体LB培养基中发酵72 h产凝乳酶的凝乳活力为(431.53±15.89)SU/mL,蛋白水解活力为(5.05±0.59)U/mL,所产凝乳酶凝乳活力高而蛋白水解活力低,凝乳酶粗酶单位酶活力为1.54×10~5SU/g。解淀粉芽孢杆菌GSBa-1是分离筛选自酒曲中的一株高产凝乳酶细菌,因此其来源安全,可作为工业化候选菌株进一步研究开发。     

牛链球菌L-(+)-乳酸脱氢酶基因的克隆及在大肠杆菌中表达

利用PCR方法从牛链球菌(Streptococcus bovis)基因组DNA中扩增出了L(+)乳酸脱氢酶基因(ldh),并将其连接到表达载体pSE380上,得到重组质粒pSEldh,将重组质粒转化到大肠杆菌菌株JM109。利用SDS-PAGE分析ldh表达产物的分子量为36KD,重组菌株的乳酸脱氢酶酶活力为168.2U/mL,最适反应温度25℃,最适作用pH为5.0,重组质粒的稳定性达99.9%。     

Purification and characterization of alkylcatechol 2,3-dioxygenase from butylphenol degradation pathway of Pseudomonas putida MT4

Alkylcatechol 2,3-dioxygenase was purified from the cell extract of recombinant Escherichia coli JM109 harboring the alkylcatechol 2,3-dioxygenase gene (bupB) cloned from the butylphenol-degrading bacterium Pseudomonas putida MT4. The purified enzyme (BupB) showed relative meta-cleavage activities for the following catechols: catechol (100%), 4-methylcatechol (572%), 4-n-butylcatechol (185%), 4-n-hexylcatechol (53%), 4-n-heptylcatechol (45%), 4-n-nonylcatechol (10%), 4-tert-butylcatechol (0%), and 3-methylcatechol (33%). The kinetic parameters, namely, K(m) and V(max), for catechol, 4-methylcatechol, and 4-n-butylcatechol, were 23.4, 8.4, and 6.5 microM and 25.8, 76.9, and 18.0 U mg(-1), respectively. These results suggest that BupB has broad substrate specificity for 4-n-alkylcatechols.     

重组极耐热内切葡聚糖酶Cel12B的诱导条件及酶的纯化

构建重组菌(JM109/pET-20b-Cel12B)生产极耐热内切葡聚糖酶,研究了不同表达宿主、IPTG诱导时间、IPTG浓度、重组菌生长不同阶段添加IPTG对重组菌生产极耐热内切葡聚糖酶的影响。结果表明:最佳诱导时间是OD值为1.0,IPTG浓度从0.5到3.0对酶活表达影响不大,诱导8h后极耐热内切葡聚糖酶表达量基本不变,优化后重组菌(E.coliBL21-CodonPlus(DE3)-RIL/pET-20b-Cel12B)表达酶活比原来菌株(E.coliJM109(DE3)/pET-20b-Cel12B)提高了53.9%,经过热处理和亲和层析得到电泳纯的蛋白条带。     

HYDROLYTIC SELECTIVITY OF PATATIN (LIPID ACYL HYDROLASE) FROM POTATO (SOLANUM TUBEROSUM L.) TUBERS TOWARD VARIOUS LIPIDS

The fatty acid and positional hydrolytic selectivity of lipid acyl hydrolase (LAH; patatin) isolated from potato tubers was determined for acylgfycerol and phospholipid substrates. LAH was about 3-fold more selective for decanoyl residues over other acyl groups of 8–18 carbons for partial glyceride substrates. For both mono- and diacylglycerols, LAH preferred substrates with primary (sn-1 (3)-) ester linkages, indicating a regiobias for these sites over sn-2-linked acyl groups. Similarly, hydrolytic activity on phospholipid substrates was 5- to 10-fold faster on sn-l-palmitoyl, sn-2-lysophospholipids than on intact phospholipids, indicating a preference for either lysophospholipids or sn-l-acyl sites, or both. LAH activity on partial glycerides was not activated by CaCl² and had a greater temperature optimum relative to LAH activity on phospholipid substrates. These differences are likely based on differences in forces and structural features conferring enzyme-substrate recognition for these substrates within a common active site.     

Purification, characterization and gene cloning of thermostable O-acetyl-L-serine sulfhydrylase forming beta-cyano-L-alanine

A thermophilic and cyanide ion-tolerant bacterium, Bacillus stearothermophilus CN3 isolated from a hot spring in Japan, was found to produce thermostable beta-cyano-L-alanine synthase. The enzyme catalyzes the synthesis of beta-cyano-L-alanine from O-acetyl-L-serine and cyanide ions. The purified enzyme has a molecular mass of approximately 70 kDa and consists of two identical subunits. It was stable in the pH range of 6.0 to 10.0 and up to 70 degrees C. The enzyme also catalyzes the synthesis of various beta-substituted-L-alanine derivatives from O-acetyl-L-serine and nucleophilic reagents. The gene encoding the beta-cyano-L-alanine synthase was isolated from B. stearothermophilus CN3. Sequence homology analysis revealed that the beta-cyano-L-alanine synthase of the bacterium is O-acetyl-L-serine sulfhydrylase. A recombinant plasmid, constructed by ligation of the cloned gene and an expression vector, pKK223-3, was introduced into E. coli JM109. The transformed E. coli cells overexpressed beta-cyano-L-alanine synthase. Heat stable beta-cyano-L-alanine synthase can be applied to the synthesis of [4-11C]l-2,4-diaminobutyric acid as a tracer for positron emission tomography.     

黄粉虫内生菌的分离鉴定及其对淀粉、纤维素和木质素的降解活性的测定

目的 分离培养黄粉虫体内细菌, 并测定这些菌对淀粉、纤维素及木质素的降解活性。方法 对黄粉虫表面消毒, 以LB和高氏一号培养基分离虫体内部细菌并进行16S rRNA基因序列分析。变色圈法测定降解活性。结果 共分离到8株细菌, 16S rRNA基因序列分析表明, 1株属于肠球菌属, 3株属于乳酸球菌属, 2株属于芽胞杆菌属, 另2株属肠杆菌属。水解酶测定结果表明, 菌株FJAT-18107具有淀粉和木质素水解作用, 其16S rRNA基因序列与解淀粉芽胞杆菌Bacillus amyloliquefaciens (NR_075005)最相似。结论 本研究为了解昆虫内生菌功能提供了参考。     

Expression of xyrA gene encoding for D-Xylose reductase of Candida tropicalis and production of xylitol in Escherichia coli

The D-Xylose reductase (XR) gene (xyrA) of Candida tropicalis IFO 0618 was expressed in Escherichia coli JM109. The enzymatic properties of each recombinant XR such as the Km value for D-xylose and NADPH, the substrate specificity for other sugars and the optimal pH were essentially the same as those of the corresponding enzyme of C. tropicalis. The recombinant XR was more heat-stable than C. tropicalis XR at 60 degrees C. E. coli, expressing the xyrA gene, successfully converted D-xylose to xylitol. When D-xylose (50 g/l) and D-glucose (5 g/l) were added to IPTG-induced cells, 13.3 g/l of xylitol was produced during 20 h of cultivation.     

Enhancement of the hydrolysis activity of β-galactosidase from Geobacillus stearothermophilus by saturation mutagenesis

Thermostable β-galactosidase (BgaB) from Geobacillus stearothermophilus is characterized by its thermoactivity in the hydrolysis of lactose to produce lactose-free milk products. However, BgaB has limited activity toward lactose. We established a method for screening evolved mutants with high hydrolysis activity based on prediction of substrate binding sites. Seven amino acid residues were identified as candidates for substrate binding to galactose. To study the hydrolysis activity of these residues, we constructed mutants by site-saturation mutagenesis of these residue sites, and each variant was screened for its hydrolysis activity. The first round of mutagenesis showed that changes in amino acid residues of Arg109, Tyr272, and Glu351 resulted in altered hydrolysis activity, including greater activity toward ortho-nitrophenyl-β-d-galactopyranoside (oNPG). The mutants R109V and R109L displayed changes in the optimum pH from 7.0 to 6.5, and the mutant R109V/L displayed different substrate affinity and catalytic efficiency (k_cat/K_m). Mutant R109G showed complete loss of BgaB enzymatic activity, suggesting that Arg109 plays a significant role in maintaining hydrolysis activity. The optimum pH of mutant E351R increased from 7.0 to 7.5 and this mutant showed a prominent increase in catalytic efficiency with oNPG and lactose as substrates.