Cloning, Purification, and Characterization of a Heterodimeric β-Galactosidase from Lactobacillus kefiranofaciens ZW3

( Xi He ),( Ning Han ),( Yan Ping Wang ) 한국미생물 · 생명공학회 2016 Journal of microbiology and biotechnology Vol.26 No.1

Lactobacillus kefiranofaciens ZW3 was obtained from kefir grains, which have high lactose hydrolytic activity. In this study, a heterodimeric LacLM-type β-galactosidase gene (lacLM) from ZW3 was isolated, which was composed of two overlapping genes, lacL (1,884 bp) and lacM (960 bp) encoding large and small subunits with calculated molecular masses of 73,620 and 35,682 Da, respectively. LacLM, LacL, and LacM were expressed in Escherichia coli BL21(DE3) and these recombinant proteins were purified and characterized. The results showed that, compared with the recombinant holoenzyme, the recombinant large subunit exhibits obviously lower thermostability and hydrolytic activity. Moreover, the optimal temperature and pH of the holoenzyme and large subunit are 60oC and 7.0, and 50oC and 8.0, respectively. However, the recombinant small subunit alone has no activity. Interestingly, the activity and thermostability of the large subunit were greatly improved after mixing it with the recombinant small subunit. Therefore, the results suggest that the small subunit might play an important role in maintaining the stability of the structure of the catalytic center located in the large subunit.

Construction and Analysis of Food-Grade Lactobacillus kefiranofaciens β-Galactosidase Overexpression System

( Xi He ),( Mingjian Luan ),( Ning Han ),( Ting Wang ),( Xiangzhong Zhao ),( Yanyan Yao ) 한국미생물생명공학회(구 한국산업미생물학회) 2021 Journal of microbiology and biotechnology Vol.31 No.4

Lactobacillus kefiranofaciens contains two types of β-galactosidase, LacLM and LacZ, belonging to different glycoside hydrolase families. The difference in function between them has been unclear so far for practical application. In this study, LacLM and LacZ from L. kefiranofaciens ATCC51647 were cloned into constitutive lactobacillal expression vector pMG36e, respectively. Furtherly, pMG36nlacs was constructed from pMG36e-lacs by replacing erythromycin with nisin as selective marker for food-grade expressing systems in Lactobacillus plantarum WCFS1, designated recombinant LacLM and LacZ respectively. The results from hydrolysis of o-nitrophenyl-β-galactopyranoside (ONPG) showed that the β-galactosidases activity of the recombinant LacLM and LacZ was 1460% and 670% higher than that of the original L. kefiranofaciens. Moreover, the lactose hydrolytic activity of recombinant LacLM was higher than that of LacZ in milk. Nevertheless, compare to LacZ, in 25% lactose solution the galacto-oligosaccharides (GOS) production of recombinant LacLM was lower. Therefore, two β-galactopyranosides could play different roles in carbohydrate metabolism of L. kefiranofaciens. In addition, the maximal growth rate of two recombinant strains were evaluated with different temperature level and nisin concentration in fermentation assay for practical purpose. The results displayed that 37oC and 20-40 U/ml nisin were the optimal fermentation conditions for the growth of recombinant β-galactosidase strains. Altogether the food-grade Expression system of recombinant β-galactosidase was feasible for applications in the food and dairy industry.

A Study of Texture Classification Based on Self-Organizing Feature Maps

Ming Yu,Xihe Shi,Huixin Yu 한국통신학회 1993 Asia-Pacific Conference on Communications Vol.2 No.2

Industrial scale extraction and stripping devices for continuous recovery of gallic acid from Chinese nutgall processing wastewater

Yundong Wu,Xihe Xia,Shuyu Dong,Kanggen Zhou 대한환경공학회 2017 Environmental Engineering Research Vol.22 No.3

In this study, we report the industrial application of an efficient technology for the recovery of gallic acid from Chinese nutgall processing wastewater. The recovery of gallic acid by industrial scale extraction and stripping devices was performed, with tributyl phosphate as the extractant and kerosene as the diluent. The results showed that the theoretical extraction stage was four, while the theoretical stripping stage was two. A closed-cycle system was studied for the continuous countercurrent extraction and stripping, with a five-stage extraction device and a three-stage reflux stripping device. The results showed that the multistage extraction-stripping system could steadily run for a long period, the average gallic acid level in the raffinate was 0.85 g·L<SUP>-1</SUP>, and the gallic acid content recovered in the strip liquor was higher than 120 g·L<SUP>-1</SUP>. The average extraction yield of gallic acid was 94.14%. When the strip liquor was used as raw material for production, the average production yield increased by 8.64%. In addition, after extraction, the CODCr in the wastewater decreased by 38.19%, and the biodegradability of wastewater improved by 1.6 times. This study provided a new impetus for the sustainable development of the Chinese nutgall processing industry.

Correlation between in vitro fertilization and artificial insemination in Holstein bulls

Sun Wei,Li Yunxia,Su Jie,Bao Xiangnan,Ding Rui,Zhao Gaoping,Cao Guifang,Hu Shuxiang,Wang Jianguo,Sun Qingyuan,Yu Haiquan,Li Xihe 아세아·태평양축산학회 2021 Animal Bioscience Vol.34 No.12

Objective: Owing to the lack of a breeding index for efficient and quick fertility evaluations of Holstein bulls when using traditional or genome-wide detection methods, this study aimed to determine whether in vitro fertilization (IVF) could be used as an indicator of conception rate of artificial insemination (AI).Methods: Conventional and sexed frozen semen from nine bulls were used for IVF and AI.Results: The IVF and AI conception rates of each bull were confirmed to be positively correlated between the conventional frozen and sexed frozen semen. The correlation coefficient R values of nine bulls between IVF and AI methods were 0.73 and 0.97 for the conventional frozen and sexed frozen semen, respectively. The average conception rate of three bulls undergoing AI was 69.5% and 64.2%, 61.8% and 58.8%, and 48.2% and 46.2% in first-, second-, and third-born cows when conventional frozen and sexed frozen semen were used, respectively, which showed a positive correlation with the fertilization rate in the same parity. We propose an evaluation standard to assess the fertilization ability of bulls based on their IVF test results, which is categorized into three grades: grade one, normal fertility bull with an AI conception rate of 40%±5% and IVF rate of 45% to 60%; grade two, higher fertility bull with an AI conception rate of 50%±5% and IVF rate of 61% to 80%; and grade three, highest fertility bull with an AI conception rate of 60%±5% and IVF rate of >80%.Conclusion: These findings reveal that IVF results can be used as a breeding index for bulls to evaluate their AI conception ability, which may shorten the time required to select bulls for breeding. Objective: Owing to the lack of a breeding index for efficient and quick fertility evaluations of Holstein bulls when using traditional or genome-wide detection methods, this study aimed to determine whether in vitro fertilization (IVF) could be used as an indicator of conception rate of artificial insemination (AI). Methods: Conventional and sexed frozen semen from nine bulls were used for IVF and AI. Results: The IVF and AI conception rates of each bull were confirmed to be positively correlated between the conventional frozen and sexed frozen semen. The correlation coefficient R values of nine bulls between IVF and AI methods were 0.73 and 0.97 for the conventional frozen and sexed frozen semen, respectively. The average conception rate of three bulls undergoing AI was 69.5% and 64.2%, 61.8% and 58.8%, and 48.2% and 46.2% in first-, second-, and third-born cows when conventional frozen and sexed frozen semen were used, respectively, which showed a positive correlation with the fertilization rate in the same parity. We propose an evaluation standard to assess the fertilization ability of bulls based on their IVF test results, which is categorized into three grades: grade one, normal fertility bull with an AI conception rate of 40%±5% and IVF rate of 45% to 60%; grade two, higher fertility bull with an AI conception rate of 50%±5% and IVF rate of 61% to 80%; and grade three, highest fertility bull with an AI conception rate of 60%±5% and IVF rate of >80%. Conclusion: These findings reveal that IVF results can be used as a breeding index for bulls to evaluate their AI conception ability, which may shorten the time required to select bulls for breeding.

Derivation of hypermethylated pluripotent embryonic stem cells with high potency

Bao, Siqin,Tang, Walfred WC,Wu, Baojiang,Kim, Shinseog,Li, Jingyun,Li, Lin,Kobayashi, Toshihiro,Lee, Caroline,Chen, Yanglin,Wei, Mengyi,Li, Shudong,Dietmann, Sabine,Tang, Fuchou,Li, Xihe,Surani, M Azi Nature Publishing Group 2018 Cell research Vol.28 No.1

<P>Naive hypomethylated embryonic pluripotent stem cells (ESCs) are developmentally closest to the preimplantation epiblast of blastocysts, with the potential to contribute to all embryonic tissues and the germline, excepting the extra-embryonic tissues in chimeric embryos. By contrast, epiblast stem cells (EpiSCs) resembling postimplantation epiblast are relatively more methylated and show a limited potential for chimerism. Here, for the first time, we reveal advanced pluripotent stem cells (ASCs), which are developmentally beyond the pluripotent cells in the inner cell mass but with higher potency than EpiSCs. Accordingly, a single ASC contributes very efficiently to the fetus, germline, yolk sac and the placental labyrinth in chimeras. Since they are developmentally more advanced, ASCs do not contribute to the trophoblast. ASCs were derived from blastocysts in two steps in a chemically defined medium supplemented with Activin A and basic fibroblast growth factor, followed by culturing in ABCL medium containing ActA, BMP4, CHIR99021 and leukemia inhibitory factor. Notably, ASCs exhibit a distinct transcriptome with the expression of both naive pluripotency genes, as well as mesodermal somatic genes; <I>Eomes, Eras, Tdgf1, Evx1, hand1, Wnt5a</I> and distinct repetitive elements. Conversion of established ESCs to ASCs is also achievable. Importantly, ASCs exhibit a stable hypermethylated epigenome and mostly intact imprints as compared to the hypomethylated inner cell mass of blastocysts and naive ESCs. Properties of ASCs suggest that they represent cells at an intermediate cellular state between the naive and primed states of pluripotency.</P>

Testicular Characteristics and the Block to Spermatogenesis in Mature Hinny

Han, Hongmei,Wang, Aihong,Liu, Liming,Zhao, Gaoping,Su, Jie,Wang, Biao,Li, Yunxia,Zhang, Jindun,Wu, Baojiang,Sun, Wei,Hu, Shuxiang,Li, Shuyu,Zhao, Lixia,Li, Xihe Asian Australasian Association of Animal Productio 2016 Animal Bioscience Vol.29 No.6

Most hinnies (female donkey${\times}$male horse) and mules (female horse${\times}$male donkey) are sterile with few reports of equine fertile hybrids. The main cause of this sterility is thought to be a meiotic block to spermatogenesis and oogenesis. This study compared the developmental features of the testes and a histological analyses of spermatogenesis in a male hinny with those of a normal, fertile stallion and Jack donkey. Hinny testes showed a thicker tunica albuginea, fewer blood vessels and more connective tissue in the testis parenchyma than those of the stallion and Jack donkey. Although the mean number of seminiferous tubules was significantly higher in stallion and hinny than Jack donkey (p<0.01), the mean proportion of seminiferous tubules was lower in the hinny (p<0.01) which resulted in a smaller diameter of seminiferous tubules. The mean number of spermatogonia and spermatocytes per unit area were significantly lower in hinny testis (p<0.01) and no spermatids or mature spermatozoa cells were found during immunofluorescent analyses. These results indicated that defects in seminiferous tubule development and structure occur in the testis of hinnies. Furthermore, most spermatogonia and spermatocytes cease development in synapsis during mid-meiosis of spermatocytes, which results in a block to spermatogenesis that prevents the formation of spermatids and matured spermatozoa during meiosis in male hinnies.