Highly efficient assimilation of lactose by a metabolically engineered strain of Saccharomyces cerevisiae

A diploid strain of Saccharomyces cerevisiae able to metabolize lactose with high efficiency has been obtained. Haploid strains of Saccharomyces able to grow on lactose were constructed by cotransformation with two genes of Kluyveromyces lactis required for the utilization of the sugar, LAC4 and LAC12, encoding β-galactosidase and lactose permease respectively. Both genes were placed under the control of a galactose-inducible promoter and targeted to the rDNA encoding region (RDN1 locus) of the Saccharomyces genome. Lac⁺ transformants were selected on medium with lactose as the only carbon source. These transformants were mitotically stable, they maintained the Lac⁺ phenotype after growing in non-selective medium for more than 60 generations, but their growth was slow. We found that this lack of vigour was caused by their genetic background and not by a deficient expression of the heterologous genes. Therefore, their performance could be improved by crossing with a wild-type strain. Among the offspring of the crosses, two strains of opposite mating type were selected and mated to obtain a fast-growing Lac⁺ diploid. This diploid strain showed the typical fermentative behaviour of S. cerevisiae when it was grown in aerated liquid medium with glucose. In lactose medium, it exhibited a respiro-fermentative metabolism similar to that of K. lactis, with low ethanol production and high biomass yield. © 1998 John Wiley & Sons, Ltd.     

EFFECTS OF β-GLUCOSIDASE ACTIVITY ON THE CHARACTERISTICS OF AROMATIC RED RICE WINE

The pigment in the bran layer of aromatic red rice (Oryza sativa var. Indica, Tapol) is rather stable, has a characteristic red color just like that of grape wine and has a peak of absorbance at 530 nm at an acidic pH. A commercial saccharifying agent, glucoamylase AN-2, produced by Aspergillus niger was fractionated to separate glucoamylase and β-glucosidase activities by column chromatography on CM Sephadex C-50. The red rice wine made from uncooked, unpolished aromatic red rice using the fractionated, β-glucosidase-free preparation of glucoamylase had a characteristic red color. By contrast, red rice wine made with glucoamylase AN-2, which contained β-glucosidase activity, was inferior in color. The red pigment of aromatic red rice wine was decolorized and glucose originating from the red pigment was released by enzymatic digestion with the fractionated preparation of β-glucosidase. The partial decolorization of aromatic red rice wine was ascribed to the enzymatic action of β-glucosidase that was present in glucoamylase AN-2. Thus β-glucosidase activity has an undesirable effect on the brewing of aromatic red rice wine.     

Characterization of a β-glucosidase isolated from an alpeorujo strain of Candida adriatica

A β-glucosidase-producing strain, Candida adriatica CECT13142, was isolated from olive oil wastes (alpeorujo) and identified by PCR/restriction fragment length polymorphism of the rDNA internal transcribed spacer and sequence analysis of the D1/D2 region of the 26S rRNA gene techniques. The enzyme was purified by sequential chromatography on DEAE-cellulose and Sephadex G-100. The relative molecular mass of the enzyme was estimated to be 50 kDa by SDS-PAGE. The hydrolytic activity of the β-glucosidase had an optimum pH of 8.2 and an optimum temperature of 40°C. The enzyme displayed high substrate specificity and high catalytic efficiency (K_m 0.85 mM, V_max 12.5 U/g of cells) for p-nitrophenyl-β-D-glucopyranoside. Although β-glucosidases have been purified and characterized from several other organisms, the C. adriatica β-glucosidase is able to have optimal activity at alkaline pH.     

Statistical optimization,partial purification,and characterization of coffee pulp β-glucosidase and its application in ethanol production

Extracellular β-glucosidase was produced using coffee pulp as a sole carbon source by Penicillium verrucosum by solid state fermentation and 897.36±59 U/g enzyme activity was obtained. Increase in 2.21-fold of enzyme activity on optimizing the bioprocess parameters by response surface methodology based on central composite rotatable design is illustrated. Maximum production level of 1,991.17 U/g was obtained with optimum values of pH 4.2, moisture 66.8%, and fermentation duration of 56 h. The enzyme was partially purified and the enzyme activity was optimum at 50°C temperature and at pH 6. The metal ions such as Mg²⁺, Zn²⁺, Ca²⁺, K⁺, detergents, and chelator such as EDTA were effective and further increased the β-glucosidase activity. On application of β-glucosidase for simultaneous saccharifiation and fermentation, 3.3% ethanol was obtained. Thus, this study provides insight on exploitation of P. verrucosum for synthesis of of β-glucosidase using coffee pulp which is available abundantly in coffee processing industries.

     

INFLUENCE OF DIFFERENT FACTORS ON ACTIVITY AND STABILITY OF ß-GALACTOSIDASE FROM LACTOBACILLUS ACIDOPHILUS1

Eight Lactobacillus acidophilus strains were tested for β-galactosidase activity. The results show that β-galactosidase is an inducible enzyme in this organism. Wide variations in enzyme properties of the strains were observed. The temperature and pH optima of the enzymes were 40–45C and 6.5–7.0, respectively. At 0.1 mM concentration, the enzyme activity in strain 4495 was slightly stimulated by 2-mercaptoethanol, partially inhibited by EDTA but remained more or less unaffected with thiomersal and ascorbic acid. In contrast, all the modulators completely inhibited the enzyme activity in strain 301. The enzyme in strain 301 was stable for 80 min at 50C, whereas the activity in strain 4495 was completely lost in 40 min. However, the enzyme was stable over a pH range of 5.8–8.0 and up to 60 days at 4 ± 1C in both strains. The K_m values for β-galactosidase from both the strains were same, whereas V_max for the enzyme from strain 301 was about 2-fold higher than that of strain 4495.     

新型碱性低分子量β-葡萄糖苷酶基因的分离与酶学性质研究

目的:获得新型β-葡萄糖苷酶编码基因,并对其表达产物进行酶学性质研究。方法:采用宏基因组学方法提取兔盲肠内微生物总DNA,构建DNA文库,通过筛选培养基获得产β-葡萄糖苷酶的克隆,测序获得其插入基因序列,氨基酸多重序列比对分析其序列特征,并用DNS显色法测定表达产物在不同条件下的酶活力。结果:在文库中获得一个基因片段nglu07,能编码一个低分子量的β-葡萄糖苷酶。nglu07基因片段长180bp,编码60个氨基酸残基。与已提交的糖苷水解酶Ⅰ家族成员进行氨基酸多重序列比对表明nglu07具有预测的活性位点Glu4与底物结合位点Tyr47,其最适反应温度为50℃,最适pH为10.0,粗酶活为8.12U/mL。结论:成功获得一新型低分子量的碱性β-葡萄糖苷酶编码基因,其蛋白温度稳定性高,在pH4.0～11.0的环境中均能保持较高的酶活力,具有作为食品工业用酶以及碱性酶的潜力,尤其在食品饮料加工、棉织品生物整理、洗涤及废纸脱墨等工业方面具有一定的应用价值。     

葡萄酒相关酵母β-葡萄糖苷酶活性及影响因素研究

以内蒙古西部地区分离到的6个属7个种共340株葡萄酒相关酵母菌株为材料,对产β-葡萄糖苷酶菌株进行初筛,进而采用对 硝基苯基-β-D-吡喃葡萄糖苷（pNPG）法检测菌株胞外、胞壁结合及胞内β-葡萄糖苷酶活力,并对高酶活性菌株中β-葡萄糖苷酶的理 化性质进行了研究。 结果表明,分属5个属5个种（葡萄汁有孢汉逊酵母（Hanseniaspora uvarum）、酿酒酵母（Saccharomyces cerevisiae）、 浅黄隐球酵母（Cryptococcus flavescens）、异常毕赤酵母（Pichia anomala）及星形假丝酵母（Candida stellata））的66株酵母菌株产β-葡萄 糖苷酶,且胞内酶活性均高于胞壁结合酶活性;不同菌株中β-葡萄糖苷酶具有相同的理化性质,4%以上葡萄糖可抑制该酶的活性, 5%～15%（V/V）的乙醇添加量对该酶活性无明显影响,其最适pH值为5.0～6.0,最适温度为40 ℃。     

酒类酒球菌β-葡萄糖苷酶研究进展

酒类酒球菌（Oenococcus oeni）是葡萄酒苹果酸乳酸发酵（MLF）中的主要微生物,糖苷物质是葡萄酒中的重要香气前体物,β-葡萄糖苷酶是降解糖苷物质的关键酶。酒类酒球菌β-葡萄糖苷酶对增加葡萄酒香气,提升葡萄酒整体品质具有重要作用。该文介绍了β-葡萄糖苷酶的定义、分类、作用机制和测定方法,阐述了酒类酒球菌β-葡萄糖苷酶活,探讨了pH值、发酵温度、乙醇浓度、糖含量和二氧化硫含量对酶活的影响,在分子生物学水平上研究了酒类酒球菌β-葡萄糖苷酶基因,并对酒类酒球菌葡萄糖苷酶未来的研究热点和研究方向进行了展望。这对深入认识葡萄酒生物增香机理和提高葡萄酒整体品质具有重要意义。     

Cloning and expression of β-glucosidases from Bifidobacterium lactis AD011

For the conversion of β-glycosides, 3 genes (BLA_0039, BLA_0141, and BLA_0893) annotated as β-glucosidase (E.C 3.2.1.21) from the genome of Bifidobacterium lactis AD011 were cloned and expressed both in Escherichia coli DH5α and Bifidobacterium bifidum BGN4 using previously established bifidobacterial expression system under the control of 16S rRNA promoter. Deduced amino acid sequence analysis revealed that BLA_0141 is highly similar to β-d-glucosidase from Bifidobacterium breve clb and is included in glycosyl hydrolase family 1 (GHF1) while BLA_0893 belongs to glycosyl hydrolase family 3 (GHF3). Recombinant BLA_0893 showed β-glucosidase activity and converted ginsenoside Rb1 to Rd. Recombinant BLA_0141 showed broad range of activities including β-glucosidase, β-galactosidase, β-xylosidase, cellobiosidase, and α-arabinopyranosidase and converted ginsenoside Rb1 and Rb2 to Rd. They also hydrolized cellobiose into glucose. The recombinant enzyme activities of both BLA_0141 and BLA_0893 were stable around pH 4.5–7.5 and below 50°C.     

产β-葡萄糖苷酶真菌的筛选鉴定、纯化及酶学性质分析

经刚果红平板染色法从土样中筛选到3株产纤维素酶的真菌,通过对其β-葡萄糖苷酶活力的测定,选择一株相对活力较高的菌株进行菌种鉴定。在形态鉴定的基础上,通过内转录间隔区(ITS)序列构建系统发育树初步鉴定为米曲霉(Aspergillus oryzae),命名为giF-10。对米曲霉giF-10进行摇瓶发酵,经硫酸铵沉淀、Sephadex G-100凝胶层析、DEAE Cellulose 52离子交换层析进行纯化。得到纯化后的β-葡萄糖苷酶,比活力为40.84U/mg,分子质量约90kD;该β-葡萄糖苷酶最适温度为55℃;在30～50℃之间热稳定性好;最适pH值为4.5;pH4.0～6.0稳定性好;金属离子对酶活力具有一定的影响,Mn2+对酶具有较强的激活作用;Fe3+和Cu2+对β-葡萄糖苷酶活力有较强的抑制作用;该酶对水杨素和纤维二糖具有较强的底物特异性;β-葡萄糖苷酶对水杨素的动力学参数：Km为0.676mmol/L;对纤维二糖的动力学参数为：Km为2.906mmol/L。     

β-glucosidase Activity in Juice-Processing Enzymes Based on Anthocyanin Analysis

A screening procedure utilizing boysenberry juice as a substrate, which combined HPLC and spectrophotometric analyses, was used to measure β-glucosidase activity of enzyme preparations used for juice processing. Enzyme preparations (n = 26) were evaluated at two dosage rates. At the mean recommended dosage, one preparation produced a decrease in total monomeric anthocyanin and cyanidin-3-glucoside, relative to a control, indicating β-glucosidase activity. At 0.1% dosage, 4 enzymes produced a significant decrease in cyanidin-3-sophoroside and 2 enzymes caused an increase in cyanidin-3-rutinoside, indicating β-1,2-glucosidase activity. Such activity in juice processing enzymes was much lower and less prevalent than found previously for β-galactosidase. Botrytis cinerea also degraded anthocyanins in boysenberry juice.     

递推式ARTP-UV复合诱变筛选高产β-葡萄糖苷酶菌株

为了提高产酶能力,采用常压室温等离子体(ARTP)与紫外(UV)复合诱变技术对酿酒酵母G13、G21菌株进行递推式复合诱变,经过一轮ARTP诱变两轮UV诱变,摇瓶培养筛选到两株高产β-葡萄糖苷酶的菌株Dea-G13D1Z2、Dma-G21D1Z2,经连续5代发酵生产试验,产酶水平可分别稳定在240.93和173.38 U/mL,是出发菌株G13、G21酶活的4.61倍和3.59倍。递推式ARTP-UV是一种有效的微生物突变育种的方法,可有效应用于微生物育种工作。     

大围山森林土壤中高产纤维素酶菌株的筛选及鉴定

通过初筛（刚果红纤维素水解试验和滤纸条分解试验）和复筛（利用3,5-二硝基水杨酸法测定内切纤维素酶活,滤纸酶活和β-葡萄糖苷酶活）,从大围山原始森林土壤中筛选高产纤维素酶菌株,获得4株具有高产纤维素酶活性的菌株。其中一株真菌16-7分解纤维素能力最强且酶活稳定,其内切羧甲基纤维素酶活为265.76U/mL,滤纸酶活为86.44 U/mL,β-葡萄糖苷酶活为39.16 U/mL;对真菌16-7分别进行形态学观察、分子生物学鉴定,初步确认为小刺青霉（Penicilliumspinulosum）。     

Description of theβ-glucosidase activity of wine yeasts

β-glucosidase activity of differentSaccharomycesstrains has been detected on the basis of its hydrolytic activity onpara-nitrophenyl-β-glucoside (pNPG) and terpene glycosides of Muscat wine. This enzymatic activity is induced by the presence of boundβ-glucose as the only carbon source in the medium and seems to be a characteristic of the yeast strain.β-glucosidase is associated with the cell wall and is found in the insoluble fraction obtained from lysed yeast cells.Saccharomyces β-glucosidase is quite glucose independent, so that its activity is reduced by about 40% in the presence of 200 g/l of glucose, but it is inhibited by about 50% with 5% ethanol in the medium; therefore, its technological use seems to be restricted to the first stages in the wine-making process.     

Designed construction of recombinant DNA at the ura3Δ0 locus in the yeast Saccharomyces cerevisiae

Recombinant DNAs are traditionally constructed using Escherichia coli plasmids. In the yeast Saccharomyces cerevisiae, chromosomal gene targeting is a common technique, implying that the yeast homologous recombination system could be applied for recombinant DNA construction. In an attempt to use a S. cerevisiae chromosome for recombinant DNA construction, we selected the single ura3Δ0 locus as a gene targeting site. By selecting this single locus, repeated recombination using the surrounding URA3 sequences can be performed. The recombination system described here has several advantages over the conventional plasmid system, as it provides a method to confirm the selection of correct recombinants because transformation of the same locus replaces the pre‐existing selection marker, resulting in the loss of the marker in successful recombinations. In addition, the constructed strains can serve as both PCR templates and hosts for preparing subsequent recombinant strains. Using this method, several yeast strains that contained selection markers, promoters, terminators and target genes at the ura3Δ0 locus were successfully generated. The system described here can potentially be applied for the construction of any recombinant DNA without the requirement for manipulations in E. coli. Interestingly, we unexpectedly found that several G/C‐rich sequences used for fusion PCR lowered gene expression when located adjacent to the start codon. Copyright © 2013 John Wiley & Sons, Ltd.     

Purification and Characterization of β-Glucosidase from Aspergillus niger

Aspergillus niger, an isolate of soil contaminated with effluents from cotton ginning mill was grown in Czapek-Dox medium containing sawdust, Triton-X 100 and urea for production of an extracellular β-glucosidase. β-Glucosidase enzyme was purified (86-fold) from culture filtrate of A. niger by employing ammonium sulphate precipitation and gel filtration on sephadex G-75. The molecular mass of the purified enzyme was estimated to be 95 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The enzyme had an optimal activity on p-nitrophenyl β-D-glucopyranoside at 50°C and pH 5.0. The K_m and V_max of the enzyme on p-nitrophenyl β-D-glucopyranoside at 50°C and pH 5 were 8.0 mM and 166 µmol/min/mg of protein, respectively. The enzyme could hydrolyze cellobiose and lactose but not sucrose. Heavy metals like Hg²⁺, Al³⁺, and Ag⁺ inhibited the activity, whereas Zn²⁺ and detergents such as Triton-X 100 and Tween-80 increased the activity at 0.01%. The enzyme activity increased in the presence of methanol and ethanol.     

Effect of enzyme treatment with β-glucosidase on antioxidant capacity of mulberry (Morus alba L.) leaf extract

This study was carried out to investigate the effect of enzyme treatment with β-glucosidase on antioxidant capacity of the mulberry leaf extract (MLE) using oxygen radical absorbance capacity (ORAC) and cellular antioxidant capacity (CAC) assay. The MLE was prepared by autoclaving at 121°C for 15 min and treated with β-glucosidase for 9 hr. High pressure liquid chromatography (HPLC) analysis showed that only qercetin-3-β-d-glucose (QT-G) among quercetin (QT) glycosides of MLE, including QT-G, quercetin-3-O-glucose-6″-acetate (QT-GA), and rutin (RT), was converted into QT by 3 hr treatment with β-glucosidase. The in vitro peroxyl radical- and hydroxyl radical scavenging capacity significantly increased after the enzyme treatment using β-glucosidase for 6 and 9 hr, respectively. The metal chelating activity increased after the enzyme treatment using β-glucosidase for 3 hr. The intracellular antioxidant capacity of MLE to protect AAPH- and Cu²⁺-induced oxidative stress in HepG2 cells significantly increased after the enzyme treatment using β-glucosidase for 3 and 6 hr, respectively, indicating that QT may be released from QT-G by β-glucosidase and penetrate into cell membrane so that it can contribute to the intracellular antioxidant capacity of MLE.     

产β-葡萄糖苷酶野生真菌的筛选鉴定及酶学性质研究

从原始森林腐木的土壤中筛选得到1株高产β-葡萄糖苷酶活力菌株Lcxs9,表型分析及ITS rDNA序列分子鉴定为芽枝霉菌,酶活为7.18U/mL,在pH值为4~9范围内,60℃以下酶活力稳定,酶谱分析芽枝霉菌Lcxs9可以产2种β-葡萄糖苷酶。该文首次报道芽枝霉菌产高活性β-葡萄糖苷酶。