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Über dieses Buch

At the ICAB 2014, researchers from around the world will gather to discuss the latest scientific research, findings and technologies concerning Microbial Genetics and Breeding, Optimization and Control of Biological Processes, Biological Separation and Biological Purification, and Advances in Biotechnology.
This conference will provide a platform for academic exchange on the application of biotechnology between domestic and international universities, research institutes, corporate experts and scholars. The participants will focus on the international development and future trends. The event will lay a solid foundation for addressing key technical challenges in various areas of applied biotechnology, providing opportunities to promote the development and expansion of the biotechnology industry.

Inhaltsverzeichnis

Frontmatter

Microbial Genetics and Breeding

Frontmatter

Chapter 1. Inheritance Analysis for Exserted Stigma Rate in Japonica Rice

Rice is the main crop in China. Since the discovery and exploitation of sterile strain line, three lines of

Indica

hybrid rice were bred successfully; the

Indica

hybrid rice has made great progress in production. Compared to

Indica

rice,

Japonica

hybrid rice is hard to study because it lacks the restorer gene. The key to promoting development of hybrid

Japonica

rice is to improve the yields of seed production. However, the low stigma exsertion rate of

Japonica

CMS Line is the main cause that restricts the yield of hybrid seed production of

Japonica

rice. As the inheritance mechanism of the stigma exsertion is complicated and difficult to study, the related study is far from enough. In this study, Jinke DS, a sterile line with higher stigma exsertion rate, and C9083, a restorer line with lower stigma exsertion rate, were used as parents for hybrid. P

1

, P

2

, F

1

and F

2

were chosen as the study materials and major gene plus polygene mixed inheritance model was chosen to analyze the genetic effects. The results showed that glume-opening single exserted stigma rate was in consistence with the E-2 model, glume-closed single exserted stigma rate was in consistence with the E-0 model, glume-opening dual exserted stigma rate, glume-opening exserted stigma rate, glume-closed dual exerted stigma rate and glume-closed exserted stigma rate were in consistence with the E-1 model.

Ruhua Wang, Na Yan, Xueyan Gao, Jie Yu, Fang Wang, Zetian Hua

Chapter 2. Cloning and Expression of β-Glucosidase from Cassava in Pichia pastoris GS115

β-Glucosidases have been widely applied in the synthesis of alkyl polyglucosides. Studies have shown that cassava β-glucosidase has great synthesis ability. This study has realized the secretion of cassava β-glucosidase gene (

mebgl

) in

Pichia pastoris

GS115. The recombinant yeast expression vector pPICZαA-MEBGL has been successfully constructed. The constructed plasmid was linearized and integrated into

P. pastoris

GS115 strain by electroporation. Positive clones were selected on YPDZ plates and then cultured in shake flask. The supernatant was collected and used for alkyl polyglucoside synthesis. The synthesis reaction was conducted under conditions: 40 °C, pH 5.0, 10 % water content, while the conditions have not been optimized. The results of these studies have indicated that 5ʹAOX promoter was the best one for expression; in the shake flask fermentation, the strain had the maximum hydrolysis activity of 60 U/L, while the optimal temperature was 35 °C, the optimal pH was 6.0,

p

NP-Glc was used as substrate; the molecular mass of the recombinant monomer protein was estimated to be 70 kDa; the recombinant protein showed high ability to transfer glucose from

p

NP-Glc to n-hexyl alcohol with high yields of 60 %, and the yields would substantially increase after optimized. This recombinant cassava β-glucosidase is expected to promote the industrialization process of alkyl polyglucoside enzymatic synthesis.

Dongheng Guo, Hongming Tian, Yanshan Xu, Suiping Zheng

Chapter 3. Construction of L-tert-Leucine Producing Strain by Expressing Heterologous Leucine Dehydrogenase and Formate Dehydrogenase in Escherichia coli

L-

tert

-Leucine is an unnatural amino acid that is a key intermediate for the synthesis of several important drugs. The L-

tert

-Leucine synthesis can be performed continuously by the collaboration of leucine dehydrogenase and formate dehydrogenase. In this study, recombinant strains of

Escherichia coli

expressing leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH), respectively, and the strain co-expressing the two enzymes were constructed. The activity for the two enzymes of the cell extraction from different recombinant strains was determined. L-

tert

-Leucine was successfully synthesized by the recombinant strains, and the yield in different conditions was compared. The production of L-

tert

-Leucine was the highest when cell extraction of strains containing pLeuDH and pFDH, 1 mL cells extract could produce 4.5 mg L-

tert

-Leucine, while 1 mL whole cells could only produce 1.05 mg L-

tert

-Leucine. The yield of L-

tert

-Leucine was 3.375 mg/mL cell extraction of the strain containing pLeuDHFDH when NAD was added, while the yield fell to 1.635 mg/mL when the whole cell was used.

Junzhen Bai, Yajian Song, Xuegang Luo, Haixu Yang, Wen Du, Tongcun Zhang

Chapter 4. The Distribution Characteristics of Microcystis novacekii Based on 16S rDNA Sequence

Microcystis

has been regarded as the most common cause of water bloom. The spatial and temporal distribution of

Microcystis

provides a method of bloom warning.

Microcystis

novacekii

was the dominant cyanobacteria species in Yuqiao reservoir in recent years. Seven sampling stations were set and the phytoplankton were collected in both July and October. Comparing morphological observation with the molecular biological method, which is based on 16S rDNA sequencing, the recognizing research of

M. novacekii was conducted

. The results showed that the average biomass of

M. novacekii

was 0.92 ± 0.23 mg/L; cell density was 1833.48 ± 457.70 × 10

4

/L affected by light, temperature, nutrients, and other environmental factors. In July, the maximum biomass was 2.21 mg/L at Center station and the minimum was 0.83 mg/L at the same site; in October, the maximum was 0.18 mg/L at intake site, the minimum was 0.09 mg/L at Center West. The significant seasonal fluctuation meant that

M. novacekii

favored high temperature, which is the same habit with universal

Microcystis

algae. 30 clones of

M. novacekii

were collected and amplified the 16S rDNA fragments. Results proved that the molecular identification of

Microcystis

, which is based on the conservative fragment of 16S rDNA, was consistent with the traditional morphology judgement, we confirmed that environmental factors have a huge impact on the distribution of

Microcystis

and the molecular biology techniques based on 16S rDNA sequencing were reliable for

Microcystis

identification.

Da Huo, Yang Luo, Yunsi Nie, Jing Sun, Yanan Wang, Zhiyi Qiao

Chapter 5. The Distribution and Molecular Identification of the Microcystis aeruginosa in Yuqiao Reservoir

Yuqiao reservoir, the only drinking water source of Tianjin in the downstream of Luan river basin, has occurrence of water bloom, at a small scale, annually. Hence the research in

Microcystis

distribution spatially and temporally has been conducted. In this study, we focus on

Microcystis aeruginosa

, which is one of the most contributing causes of the bloom. Samples are collected from seven sites in July and October 2012 and molecular techniques conducted to identify the selected clone from the samples by 16S rDNA sequencing. According to the results,

M. aeruginosa

has grown vigorously in July with the average biomass of 1.59 ± 0.39 mg/L and the cell density of 3191.25 ± 768.04 × 10

4

/L. The maximum value occurs in the site of the northern reservoir (the average biomass and the cell density were 1.37 ± 1.07 mg/L and 2723.33 ± 2131.13 × 10

4

/L respectively). By analyzing the sequencing results and comparing with the morphological characters, the accuracy of molecular identification based on the 16S rDNA region is confirmed. From the BLAST alignments in NCBI, we find that the homology of 30 clones of

M. aeruginosa

is between 97 and 99 %; A+T (56.6 %), which is higher than C+G (43.4 %). The samples almost match the same branch of phylogenetic tree.

Zhiyi Qiao, Da Huo, Jun Zhang, Jing Sun, Yunsi Nie, Yanan Wang

Chapter 6. The Improved Stress Tolerance of Escherichia coli by Directed Evolution of IrrE

IrrE, a global regulator of

Deinococcus radiodurans

, has proven to be effective in enhancing microbial stress tolerance. In this paper, IrrE from

D. radiodurans

R1, was introduced into

Escherichia coli

and directed evolved by error-prone PCR. The influence of mutation of IrrE on the cell growth and tolerance to various stresses was further investigated. First, one of the mutations, designated M4 with higher ethanol tolerance was obtained by error-prone PCR using the pET-28a(+)-

irrE

(W) as a template. The OD

600

value of M4 reached 1.6 after 28 h cultivation under 8 % ethanol, while no obvious cell growth was observed in the recombinant strain harboring plasmid pET-28a(+)-

irrE

(W) and the control strain harboring plasmid pET-28a(+). The cell viability of M4 under different stress shock conditions (such as pH = 5.0, pH = 10.0, 3 mmol/L sorbitol and 15 % methanol), the cell growth rate, and the final biomass were improved obviously. The sequence comparison of

irrE

revealed that there were two sense substitutions (C24T and G530A). The substitution of G530A caused one amino acid change Gly177Glu. The homology modeling of IrrE was built according to the known structure of IrrE protein from

D. deserti

, which showed that the amino acid mutation located in HTH motif of IrrE. The work laid a foundation for further research on the relationship of IrrE structure and host cell tolerance to stress.

Jianmei Luo, Jiajia Liu, Yuanyuan Zheng, Min Wang

Chapter 7. Cloning and Characterization of the TMEPAI Gene Promoter

TMEPAI

was originally identified as a highly androgen-induced gene by serial analysis of gene expression in androgen-treated prostate cancer cell.

TMEPAI

is highly expressed in many tumor cells. However, little is known about the transcriptional mechanism regulating

TMEPAI

gene expression, and its promoter has not yet been characterized. In this study, the 5ʹ regulatory region of the

TMEPAI

gene was characterized, the 5ʹ flanking sequence of

TMEPAI

gene was successfully amplified by PCR, and the 925 bp fragment of

TMEPAI

gene promoter was inserted into pGL4-Basic vector to measure its activity. Furthermore, the transcriptional regulators in

TMEPAI

promoter region were analyzed by bioinformatics. Taken together, these results will help to understand the expression regulation of

TMEPAI

and its role in tumorigenesis.

Ailong Guo, Ping Qin, Weiwei Shi, Yuyin Li, Aipo Diao

Chapter 8. Cloning and Expression of a Novel Xylanase Xyn11-1 from Alkaline Soil

A novel xylanase of family 11 (

Xyn11

-

1

) was obtained from the metagenomic DNA of alkaline soil by touchdown-PCR and thermal asymmetric interlaced (TAIL) PCR methods.

Xyn11

-

1

is composed of 645 nucleotides, which encodes a signal peptide of 19 amino acids and a mature protein of 196 amino acids. It is a novel GH11 xylanase sharing the highest identity (79 %) with the reported GH11 xylanase (XP_008721536) in GenBank database. In order to detect its biological activity, the recombinant plasmid

xyn11

-

1

-pET28a(+) was constructed and recombinant protein was successfully expressed in heterologous hosts

Escherichia coli

BL21 (DE3) induced by isopropy-beta-

d

-thiogalactopyranoside (IPTG). Using the 3,5-dinitrosalicylic acid (DNS) method, the xylanase activity of crude intracellular protein is 11.32 U/mL. The optimal inducing IPTG concentration and inducing temperature was examined at 0.2 mM and 15 °C, respectively.

Kun Li, Zhongyuan Li, Xuegang Luo, Cuixia Feng, Cuiqiong Wang, Minghui Zhang, Tongcun Zhang

Chapter 9. To Establish the Regeneration System of Sweet Sorghum Immature Embryos

Sweet sorghum is an important forage and energy crop. In this experiment, sweet sorghum immature embryos were taken to be explants, through inducting and regenerating for sweet sorghum callus of different genotypes, to obtain the best culture medium formula and create appropriate conditions for establishing efficient regeneration system. The result showed that, for callus induction, the response of immature embryos varied with the genotype, but the medium is not. Of these, Xinliang 52 showed the highest frequency of callus induction under appropriate culture conditions. As regards regenerable callus formation, the response of callus varied with the genotype and medium. 07-27 regeneration occurred at high frequencies when MSR and MBR were added in the regeneration medium. And there is nothing on NBI medium for the whole genotypes. So NBI is not suitable for callus regeneration culture.

Xiaomu Chen, Oujing Li, Lili Shi, Xianhua Wu, Boxian Xia, Zhongyou Pei

Chapter 10. Cloning and Sequence Analysis of a Novel ACC Oxidase Gene from Peanut (Arachis hypogaea L.)

Ethylene plays important roles in seed dormancy. As a key enzyme in ethylene biosynthesis, ACC oxidase gene (

ACCO

) converts 1-aminocyclopropane-1-carboxylic acid (ACC) into ethylene. To study the mechanism underlying peanut seed dormancy, cDNA sequence of the candidate gene was isolated from peanut using RACE technique. As in the situation in

Arabidopsis

, the

ACCO

from peanut (

AhACCO

gene) contained four exons and three introns. Homology comparison analysis showed 68–87 % sequence similarity between

AhACCO

and

ACCO

genes of other selected plants.

Quanxi Sun, Xiuzhen Wang, Yueyi Tang, Qi Wu, Yunyun Wang, Qingyun Zhang, Guangying Cao, Shuo Meng, Chuantang Wang

Chapter 11. Genetic Diversity Among the Microorganisms in Daqu Used for Beidacang Liquor as Revealed by RAPD Analyses

To assess the genetic diversity among five microorganisms (namely, DSL(CS)-01, DSL(CS)-02, DSL(PDA)-C, DSL(PDA)-E, DSL(PDA)-F), RAPD (random amplified polymorphic DNA) analysis was performed. Seven primers with stable polymorphisms were screened from 64 primers. PCR was carried out with the seven primers. The data indicated that the molecular markers gave different levels of polymorphism. 94 RAPD bands were obtained and out of them 64 were polymorphic bands (68.09 %) and the best primer screened was b8. The primer b8 was considered as the most effective RAPD marker for the five microorganisms above. The dendrogram built on the basis of data from RAPD analysis represented the genetic distances among the five microorganisms. Understanding the genetic variability among the microorganisms opens up a possibility for developing a molecular genetic map that will lead to the application of marker-assisted selection tools in genetic improvement of the microorganisms.

Shi-Wei Wang, Qing-Hui Wang, Li-Ping Zhai, Jun Liu, Zhi-Dan Yu, Mao-Mao Zheng, Fang Wang

Chapter 12. Stenotrophomonas maltophilia Having Decolorization Capability of Azo Dye Isolated from Anaerobic Sludge

Strain 5-8 was isolated from anaerobic sludge which was collected from a sewage treatment plant located at Shandong Province. Strain 5-8 has efficient ability to decolorize Fast Yellow2G. Fast Yellow2G was decolorized by more than 80 % in 48 h, and completely decolorized in 60 h. Strain 5-8 was observed to form yellow colored, smooth, circular, convex colonies of 5 mm diameter with entire margins on LB medium. Cells of strain 5-8 were observed to be gram-negative, aerobic, motile with a single polar flagellum and rod-shaped with dimensions of 0.3–0.4 × 0.6–1.5 µm. Growth was observed at 18–40 °C (optimum 35–37 °C), pH 6.0–9.0 (optimum pH 7.0–8.0) and with 0.5–5 % (w/v) NaCl (optimum 1 %). Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showed that strain 5-8 belongs to the genus

Stenotrophomonas

, clustering coherently with the type strain of

Stenotrophomonas maltophilia

showing sequence similarity value of 99 %.

Wei Feng, Peng Song, Yang Zhang, Zixing Dong

Chapter 13. In Silico Cloning and Sequence Analysis of F3H Gene in Raphanus sativus L.

Flavanone 3-hydroxylase is a key enzyme in the biosynthetic pathway of plant flavonoids. The complete cDNA sequence of

Raphanus sativus

L. F3H gene was in silico cloned using

Brassica napus

F3H-2 gene cDNA sequence (DQ513329.1) as a probe. Then the hydrophilicity, secondary structure, and advanced structure of F3H protein in

R. sativus

L. were analyzed using bioinformatics methods. The results showed that the cDNA was 1,437 bp, contains an open reading frame of 1,077 bp, encoding 358 amino acids. The protein coded by

R. sativus

L. F3H gene cDNA showed 98 % similarity to

B. napus

F3H-2. Its secondary structure contains 38.55 % alpha helix, 41.9 % random coil and 4.93 % beta turn. The homology analysis of 3D structure showed that the three-dimensional structure of the protein is a compact globular structure.

Guang Ma, Jiping Guo

Chapter 14. The Comparative Study of Fermentation Capability of Wheat Beer Yeast

In this study, the wheat beer fermentation performances of three beer yeasts S-15, S-16 and S-17 were comparatively studied. Here the best strain was selected for fermentation of wheat beer. Comparison of three strains had been experimented at fermentation capability, such as sugar-reducing rate, higher ethanol concentration, fermentation degree, agglutinating, and alcohol yield. Among these three yeasts, sugar-reducing rate of S-17 was the quickest and up to 0.1452 Brix/h. The data showed that S-17 could rapidly utilize sugar to ferment and was better in the brewing process to initiate fermentation. After 96 h of fermentation, alcohol yield of S-17 was slightly higher than other yeasts in agreement with sugar-reducing rate and up to 3.55 % (v/v). The real fermentation degree and the apparent fermentation degree of S-17 were also higher than the other two yeasts. Three beer yeasts belong to weak yeast coherency and the sequence of yeast coherency was S-17 > S-16 > S-15. The total content of high alcohol for S-17 was 126.39 mg/L, in which, the main ingredient is isopentanol and up to 56.48 %. The content of the high alcohol produced by S-17 during beer fermentation is in the normal range. Under the same conditions, strain S-17 showed faster fermentation speed, shorter fermentation period, higher alcohol degree, and stronger cohesion property. So S-17 was selected for wheat beer fermentation.

Jinxu Sun, Yue Wang, Cuicui Wang

Chapter 15. Screening of Dual Defects Strain and Effects on l-Isoleucine Production in Escherichia coli NML

Original strain

Escherichia coli

N12 with lifting the repression of

l

-isoleucine was mutated mutagenesis by diethylsulfate (DES), then

l

-isoleucine producing strain named

E. coli

NML were obtained. The effects of

E. coli

K12, N12, and NML in fermentation process on biomass, yield of

l

-isoleucine and glucose consumption rate were studied by carrying out fed-batch fermentation by analyzing the metabolic sysnthesis process. At 48 h,

l

-isoleucine production in the fermentation medium of N12 and NML was 2.25 and 4.64 g/L, respectively, and it was not detected in

E. coli

K12. Results show that mutant strains with dual genetic markers (Met

+ Leu

) can help with the synthesis of

l

-isoleucine, which also provide some frame of reference for the production of other amino acids.

Linan Yu, Huiyan Liu, Haitian Fang, Qing Wu

Chapter 16. Effect of MIG1 Gene Deletion on Lactose Utilization in Lac+ Saccharomyces cerevisiae Engineering Strains

A lactose-consuming

Saccharomyces cerevisiae

strain EY-510 was constructed by expressing

LAC4

and

LAC12

gene of

Kluyveromyces marxianus

in the host strain AY-5.

Mig1

is a zinc finger DNA-binding protein that plays a critical role on glucose repression in

S. cerevisiae

. In order to study in anaerobic condition the degree of glucose repressing galactose metabolism, a deletion fragment

MIGIA

-

KanMX

-

MIG1B

was transformed into AY-5, resulting a Δ

mig1

strain DY-510. In order to study whether the presence of glucose inhibits the consumption of lactose, a Lac

+

Δ

mig1

strain RY-510 was constructed by transforming the deletion fragment into EY-510. Galactose consumption was initiated at higher glucose concentrations in the

MIG1

deletion strain RY-510 and DY-510 than in the corresponding wild-type strain AY-5 and EY-510, wherein galactose was consumed until glucose was completely depleted in the mixture. On lactose medium, the duration of fermentation for RY-510 was 168 h, whereas the duration for EY-510 was 252 h. The lactose uptake rate was 0.357 g/L/h for RY-510 and that was 0.238 g/L/h for EY-510. The ethanol productivity of RY-510 was 0.127 g/L/h and that was 0.085 g/L/h for EY-510. And in cheese whey powder solution medium, RY-510 was able to produce 30.25 g/L ethanol from 76.8 g/L initial lactose in 190 h, during which EY-510 was able to consume 70.8 % of the initial lactose and produced 24.14 g/L ethanol. Therefore, relieving glucose control provides an approach for constructing lactose-consuming

S.

cerevisiae

.

Jing Zou, Xuewu Guo, Jian Dong, Cuiying Zhang, Dongguang Xiao

Chapter 17. Enzyme Activity Analysis of Protease Produced by Marine Bacteria

Proteolytic enzymes are ubiquitous in nature, found in all living organisms, and have been applied to many aspects of industry. In this study, 19 strains hydrolyzing protein were screened from deep-sea sediments, of which No. 9 strain has the largest protease activity. Based on morphological characterization and 16S rDNA gene sequences analysis, the strain was identified to be

Bacillus subtilis

. The optimum medium for producing protease was determined through orthogonal test as follows: 2.5 % xylose, 3.5 % peptone, 1.6 % yeast extract and 1 % NaCl, the highest protease activity reached 215.95 U/mL, and increased by 128.06 % than the origin.

Qi Zhang, Xihong He, Hao Liu

Chapter 18. Expression of Gene uvrA from Acetobacter pasteurianus and Its Tolerance to Acetic Acid in Escherichia coli

The

uvrA

gene of

Acetobacter pasteurianus

AC2005 coding for subunit A of the excinuclease ABC complex involved in the nucleotide excision repair mechanism was identified. Gene

uvrA

was amplified using

A. pasteurianus

AC2005 genomic DNA as a template. Then the pMV24 plasmid, an expression vector of

Acetobacter

, was used for constructing the recombinant plasmid pMV24-

uvrA

. UvrA was expressed in

Escherichia coli

JM109, and its molecular weight was about 91.1 kDa. With 0.5 % acetic acid shock for 20 and 40 min, the survival rates of recombinant strain

E. coli

JM109/pMV24-

uvrA

were 0.48 and 0.056 %, which increased by 17.5 and 10.2 times, respectively, compared with those of

E. coli

JM109/pMV24. All these demonstrate that the expression of repair excinuclease UvrA could increase the acetic acid tolerance of the strain.

Yu Zheng, Xingjing Chen, Jing Wang, Haisong Yin, Liqing Wang, Min Wang

Chapter 19. Construction of Eschericha coli-Staphylococcus Shuttle Vector for EGFP Expression and Potential Secretion via Tat Pathway

In this study, the potential for heterologous protein expression and secretion via twin-arginine translocation (Tat) pathway was investigated in

Escherichia coli

DH5α host using enhanced green fluorescent protein (EGFP) as model protein reporter. To construct the shuttle vector pBT2-ET-5X-EGFP, 17 kinds of PCR-amplified

5x

-

egfp

fragments were, respectively, cloned into plasmid pBT2-Peftu-Tat-EGFP and transformed into

E. coli

DH5α host. By SDS-PAGE, fluorescence microscope observation and flow cytometry analyzation, EGFP was expressed in an active form in the cells of

E. coli

DH5α, but failed to translocate to the culture medium.

Bao-yin Xu, Yi-bing Cheng, Lin Wang, Hao Zhou, Lin Huang, Xiao-yan Tang, Qiang Gao

Chapter 20. SNP Affects the Mobility of Breast Cancer Cells and the Expression of Metastasis-Related Genes

Protein S-nitrosylation is a type of posttranslational modification that changes protein stability, intracellular localization, and biological activity. In mammalian cells, NO is synthesized by NOS which is highly expressed in many solid tumors, including breast cancer. NOS is involved in the progress of carcinogenesis by generating NO to modify various tumor-related proteins to enhance or suppress their function. However, the effect of NO on tumor metastasis is less studied. Herein, breast cancer cells MCF-7 and MDA-MB-231 were treated with NO donor SNP, which resulted in altered cell mobility. Meanwhile the expression of several metastasis-related genes was regulated. These results suggest a novel mechanism by which NO promotes the metastasis of breast cancer.

Juan Hu, Hongpeng He, Hao Zhou, Dandan Wang, Yijie Wang, Xuena Liu, Yongwei Lai, Tongcun Zhang

Chapter 21. Molecular Cloning and Characterization of Glycerol Dehydrogenase from Klebsiella pneumoniae

Glycerol dehydrogenase (GDH) was a key enzyme for 1,3-propanediol fermentation from glycerol. In this report, gene

gdh

, encoding GDH of

Klebsiella pneumoniae

KG1 was gained by PCR with genomic DNA as template. The open reading frame (ORF) of

gdh

consisted of 1,143 bp and encoded 380 amino acids with a deduced molecular mass of 42 kDa. The gene

gdh

was overexpressed in

E. coli

BL21(DE3) and produced 886-fold higher activity of GDH than that of KG1. The optimum temperature of recombinant GDH was 37 °C and glycerol was the optimum substrate.

Yanhua Liu, Li Zhao, Jianguo Zhang, Yu Zheng

Chapter 22. Expression of Glucose-6-Phosphate Dehydrogenase and 6-Phosphogluconate Dehydrogenase Improve l-Citrulline Biosynthesis in argG-Deleted Corynebacterium glutamicum

Corynebacterium glutamicum

, a well-known producer in the amino acids industry, has become a potential platform organism for synthetic biology in industrial biotechnology. During

l

-citrulline biosynthesis, NADPH is required as a crucial cofactor. Production of

l

-citrulline requires 2 mol of NADPH per mole of

l

-citrulline. The strategy focused on engineering of the pentose phosphate pathway (PPP) flux by different genetic modifications. In this study, two NADPH-supplying strategies based on glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was compared, and their influences on

l

-citrulline biosynthesis were examined. The

zwf

and

gnd

genes were overexpressed in the

l

-citrulline-producing strain CgΔargG. The expression of both genes greatly enhanced intracellular NADPH concentration and

l

-citrulline production. The concentration of intracellular NADPH was increased by 287 and 363 %, and the production of

l

-citrulline was increased by 30.8 and 20.5 % in CgΔargG/pXMJ19-zwf and CgΔargG/pXMJ19-gnd, respectively, compared with CgΔargG. The concentration of NADPH in a

zwf

- and

gnd

-expressing strain was increased by 287 and 363 %, respectively. These results are helpful for improving

l

-citrulline biosynthesis and other biosynthetic processes.

Zhaoxing Liu, Luping Chen, Ning Hao, Lin Xu, Yan Li, Ming Yan, Pingkai Ouyang

Optimization and Control of Biological Process

Frontmatter

Chapter 23. Production of Amino Acids by Mixed Bacterial Strains-Mediated Solid State Fermentation of Feathers and Dynamic Changes to the Fermentation System

Bioprocessing of chicken feather wastes for production of free amino acids was investigated by solid-state fermentation (SSF) with (1) single strain, (2) double strains, (3) triple strains of

B. licheniformis

TCCC 11593,

B. alcalophilus

TCCC 11004 and

B. subtilis

TCCC 11279. The maximum production of total amino acids amounting to 161.35 mg/g was obtained under optimal SSF conditions: feather concentrations 1.0 g/250 mL, moisture contents 1:9 (w/v), inocula ratios (v/v) of 5:5:3 for

B. licheniformis

:

B. alcalophilus

:

B. subtilis respectively

, 60 h fermentation. Monitoring of mixed bacterial populations in the SSF process by real-time PCR indicated that

B. alcalophilus

was the dominant microorganism present in the SSF cultures. Moreover, measurements of dynamic changes in multienzyme activities in the SSF process suggested that, in terms of amino acid production, keratinase might play an important role in the initial hydrolysis of keratin and the further hydrolysis was most likely to be accomplished by elastase, collagenase, alkaline protease, and neutral protease elaborated by the three mixed bacterial strains. The above results indicated that mixed-strain SSF could serve as a cost-effective method to utilize chicken feather wastes to produce value-added feed additives or organic fertilizers.

Yu Li, Dunji Hu, Sheng Chen, Xiangnan Lei, Xiangjin Zhang, Xiaoguang Liu, Fuping Lu

Chapter 24. Effects of Culture Medium on PUFAs Production by Mortierella isabellinas

In the paper, in order to improve the polyunsaturated fatty acids (PUFAs) by

Mortierella isabellina

, the effects of carbon source, nitrogen source, carbon to nitrogen (C/N) ratio, and metal ions on PUFAs production were investigated. The results of the research showed that different kinds of carbon source and nitrogen source had obviously effect on PUFAs production, the glucose and mixture nitrogen (yeast extract: peanut meal at 3:1 (w/w)) were greatly suitable for PUFAs production and cell growth. The glucose concentration and C/N ratios have significant effect on PUFAs production. Meanwhile, the results indicate the metal ions have profound effects on fungus growth and PUFAs production. Ca

2+

and Mg

2+

could promote PUFAs production. Almost all the improvement of PUFAs was obtained through increasing thallus biomass, though there were some influences on the metabolism. Under the optimal culture media composition, the yields of cell dry weight, linoleic acid (LA), gama-linolenic acid (GLA), and total PUFAs were 32.48, 2.90, 2.83, and 7.36 g/L, respectively.

Dengyue Sun, Cuixia Zhou, Chuanhe Zhu, Yanru Sun

Chapter 25. A Simple Mechanochemical Cycle Model for Dynein

Dynein is the largest and most morphologically complex of biological molecular motors. By analyzing and summarizing the structure and experimental parameters, we develop a mechanochemical cycle model for dynein hydrolyzing ATP. Based on the model, we discuss the relationship between the load and the step size of the dynein, the result derived fit well with the experimental results.

Xiaoyang Zhao, Wei Sun, Junping Zhang, Tala Lei, Weisheng Guo

Chapter 26. The Research on Biotransformation Pathway of Digitoxin by Aspergillus ochraceus and the Analysis of Products Activity

In order to analyze the biotransformation pathway of digitoxin by

Aspergillus ochraceus

Rn405, a product—digitoxigenin was used as the substrate to biotransform. The substrate consumption and product synthesis were monitored by HPLC during the bioconversion process. The preliminary results were as follows: Firstly, hydrolysis reaction happened and digitoxin was converted into digitoxigenin; then, with extension of the reaction time, the hydroxyl group was introduced to the C-11 of digitoxigenin by the role of hydroxylase and another product named 11α-hydroxydigitoxigenin was generated in a large amount. The influences of these two products on myocardial apoptosis and their antineoplastic activities were further analyzed. It was found that compared with the substrate digitoxin and reference substance digoxin, the apoptosis effects of digitoxigenin and 11α-hydroxydigitoxigenin on rat myocardial cells remarkably decreased; but no obvious antineoplastic activities on the four tumor cells, including human breast cancer cell MCF-7, rat breast cancer cell 4T1, human cervical cancer cell Hela, and rat melanoma cell B-16 were observed.

Jianmei Luo, Ting Song, Fangfang Cui, Yanbing Shen, Min Wang

Chapter 27. Astragalus membranaceus Polysaccharide-Enhanced Lymphocytes Proliferation of Yellow Drum Nibea albiflora In Vitro

To quickly evaluate the immunostimulatory effect of

Astragalus membranaceus

polysaccharide (AMP) on yellow drum

Nibea albiflora

, the head kidney lymphocytes proliferation was tested with/without concanavalin A (Con A) or lipopolysaccharide (LPS) in vitro. The lymphocytes were incubated within 0, 25, 50, 100, 200, 400, and 800 µg/mL AMP for 48 h, with/without Con A (2.3 µg/mL) or LPS (18.2 µg/mL). Results showed that there was a dose-dependent relationship between lymphocytes proliferation and the AMP concentration (

P

< 0.0

5

). The AMP-enhanced lymphocytes proliferation at 50–200 µg/mL significantly (

P

< 0.05), with peak value at 100 µg/mL. Low dose (0–25 µg/mL) and high dose (400–800 µg/mL) of AMP did not stimulate lymphocytes proliferation significantly (

P

< 0.05). Synergetic effects were observed between AMP and Con A/LPS on lymphocytes proliferation. Data in this chapter implied that the AMP might contribute to both cellular and humoral immunity of

N. albiflora

in a dose-dependent manner.

Huilai Shi, Fangping Yu, Qingkui Wang

Chapter 28. Aspergillus niger Pellets Absorbed Bacillus sp. Isolated from Soybean Wastewater Sludge

More efficient soybean wastewater treatment approach is necessary because of the increasing soybean processed in the worldwide.

Aspergillus niger

pellets formed during the wastewater treatment was a promising method from the views of easy harvest, safety authorized by FDA, and lower sludge obtained. For further COD removal on the base of

A. niger

pellets, a bacteria (

Bacillus

sp.) was isolated from sludge of classical soybean wastewater treatment. And then the absorption ratio was tested in different conditions for potential application in wastewater treatment. In this research, 76.24 % of

Bacillus

sp. was absorbed from the 3.0 × 10

8

/mL initial cell concentration at pH 5.0. These results confirmed the possibility of further COD removal by the combination of

A. niger

and bacteria without losing the advantages of fungal pellets.

Ningning Diao, Xiaowei Wu, Jianguo Zhang

Chapter 29. Expression, Purification and Characterization of Maltase from “Quick” Baker’s Yeast

The “quick” baker’s yeasts are capable to rapidly metabolize maltose, thereby improving leavening ability. Given that maltase is the determining factor in maltose fermentation for “quick” baker’s yeast, it is hence necessary to research the properties of the maltase independently for the “quick” baker’s yeast. In this study, the heterogeneous expression of MAL62 encoding for maltase and purification of maltase were well completed. Furthermore, the enzymatic properties of maltase concentrated on the effects of substrate (maltose) and end product (glucose) were investigated as well as the physic-chemical properties and transglycosylation activity. The substrate maltose did not affect the activity of maltase, while the maltase activity was inhibited by the end product. This study provides guidance for the research of maltose metabolism for “quick” baker’s yeast.

Cui-Ying Zhang, Hai-Yan Song, Xue Lin, Xiao-Wen Bai, Dong-Guang Xiao

Chapter 30. Evaluation of an Ethanol-Tolerant Acetobacter pasteurianus Mutant Generated by a New Atmospheric and Room Temperature Plasma (ARTP)

A novel atmospheric and room temperature plasma (ARTP) which used helium as the working gas was employed to generate mutants of

Acetobacter pasteurianus

to improve the ethanol tolerance, which is a poorly characterized industrial strain. The best strain U1-1 was selected after mutagenesis. U1-1 could grow in liquid medium with 11 % ethanol. The production of acetic acid reached 32.83 ± 0.75 g/L, 385.7 % higher than that of the parent strain, meanwhile, U1-1 has stable production. Moreover, the cell membrane permeability were measured by PI assay and the results show that the cell membrane permeability of starting strain (AP-1.01) is significantly higher than U1-1. The better ethanol tolerance of strain U1-1 was maybe due to the decreased membrane permeability.

Xiaoying Wu, Yuqiao Wei, Zeming Xu, Lingpu Liu, Zhilei Tan, Shiru Jia

Chapter 31. A Thermotolerant Acetobacter pasteurianus T24 Achieving Acetic Acid Fermentation at High Temperature in Self-Adaption Experiment

The aim of the present work was to improve the thermotolerance of acetic acid bacteria. In this study, a thermotolerant

Acetobacter pasteurianus

T24 was obtained through adaptive experiments. The strain T24 exhibited better growth at 40 °C in solid medium. The strain T24 exhibited rapid growth and the wild stain HN101 exhibited a longer lag phase at 40 °C in acetic acid fermentation than strain T24. Under the condition of low ethanol concentration, the highest acetic acid concentration produced by T24 at 40 °C increased by 18.05 % over the wild stain HN101. These results indicated that the adapted strain had acquired thermotolerance over the course of adaptation. Thus, this strain was used for acetic acid fermentation at high temperature. This work reveals the potential value for improvement in industrial vinegar production.

Yuqiao Wei, Xiaoying Wu, Zeming Xu, Zhilei Tan, Shiru Jia

Chapter 32. Optimization of Cultural Conditions for Extracellular Polymeric Substances (EPS) Production by Burkholderia Using Response Surface Methodology

Extracellular polymeric substances (EPS) are complex biopolymers secreted by a variety of microorganisms. Except for the basic protecting cells from rigorous environment, EPS also influenced the bioflocculation and settleability of sludge. There were reports about the application of EPS in minimizing harbor siltation. On the basis of this point, a series of experiment were proceeded. Based on single factor experiment, the cultural conditions for EPS production were investigated and optimized by response surface methodology (RSM) at the Box–Behnken design. Optimal EPS yield (6.40 g/L) was obtained at a combination of sucrose 21.0 g/L, NH

4

Cl 1.1 g/L, MgSO

4

0.2 g/L. And the influence of NH

4

Cl for EPS yield was the most significant, as the rest in order was sucrose and MgSO

4

. A verification experiment confirmed the reliability of the model.

Baojiang Sun, Peipei Han, Ruyu Tao, Qixiu Pang, Shiru Jia

Chapter 33. Effects of pH, Temperature, Storage Time, and Protective Agents on Nisin Antibacterial Stability

Nisin as a kind of bacteriocin and peptides can inhibit the growth of gram-positive bacteria effectively, which is easily influenced by heat, alkali, and storage time significantly. In this study, it is concluded that adding substances such as chitosan, ascorbic acid, tyrosine, and FeSO

4

do well in the enhancement of nisin antibacterial stability. The protective agents have been proposed to improve stability and long-term effectiveness of nisin. Above all chitosan was of great efficiency, followed by FeSO

4

and tyrosine, but ascorbic acid was better in acidic conditions. Similar phenomenon occurred when nisin was at elevated pH and high temperature. At pH 6.0, room temperature, nisin titer got rise to 154.7 IU/mL after added chitosan compared to the original 105.6 IU/mL, and even after the 121 °C heating for 20 min, the titer was 2.8 times higher than control.

Zhilei Tan, Jing Luo, Fang Liu, Qian Zhang, Shiru Jia

Chapter 34. Enhanced Solvent-Stable Alpha Glycosidase Production by Bacillus licheniformis JXC-1 by Optimization of Feeding Strategies

In the previous work, we screened a strain (

Bacillus licheniformis

JXC-1) producing solvent-stable (10 %

N

,

N

-Dimethylformamide, short for DMF) α-glucosidase from several different soil samples. In this work, we attempt to improve the α-glucosidase production by optimization of feeding strategies in a 3-L fermenter. Specifically, the key factors of solvent-tolerant α-glycosidase production were investigated first, and the optimal conditions (pH 7.0, the initial maltose concentration 25 g/L, and agitation speed 600 rpm) were obtained; the enzyme activity reached 444.7 U/L under the optimal conditions. Then, four feeding strategies with different feeding rates for 4–8 h to feed maltose or both maltose and tryptone were carried out, and it was found that feeding maltose and tryptone at a rate of 2.25 mL/h (4–5 h), 6.75 mL/h (5–6 h), 9 mL/h (6–7 h), and 15 mL/h (7–8 h) significantly increased the α-glycosidase production, from 444.7 to 872.5 U/L.

Jun Fang, Qunfang Tang, Long Liu, Jianghua Li

Chapter 35. The Effect of Different Activated Carbon and Bleaching Temperature on Kojic Acid Bleaching

In this paper, the effects of six different types of activated carbons on decolorization of kojic were studied and two kinds of them were picked out based on the decoloration rate or kojic acid yield, respectively. After mixing in different proportions, the effects of different ratios of the two activated carbons at different bleaching temperature conditions on kojic bleaching were determined by experiments. The results show that the decoloration rate was the highest (79.84 %) at 70 °C when the ratio of 767 was 20 % (total 0.2 g of activated carbon), and it had a higher yield of kojic acid (91.26 %), while the highest kojic acid rate (94.06 %) was obtained at 80 °C when the ratio of 767 was 100 %; the decoloration rate was also better (73.26 %) under these conditions. In contrast, the better bleaching effect with minimum energy consumption was at 50 °C when the ratio of 767 was 40 %, the kojic acid yield reached more than 90 %. These three combined ratios have their own advantages, according to the different needs for various occasions.

Wu Meng, Cuiying Zhang, Dongguang Xiao

Chapter 36. Effect of Iodine on the Growth and Quality of Nostoc flagelliforme

This study was carried out to evaluate the effect of diverse iodine form-iodide and iodate on the biomass as well as the content of soluble protein, pigment, and iodine in

Nostoc flagelliforme

cells. In our study, tested factors (iodine form-iodide and iodate) significantly influenced the growth and content of soluble protein, pigment, and iodine in

N. flagelliforme

cells. Moreover, results obtained have shown that

N. flagelliforme

cultured with I

take up greater amounts of iodine than those treated with IO

3

. Besides, at the initial iodine concentration of 600 mg/L in the form of KI and KIO

3

, the biomass of

N. flagelliforme

significantly increased by 2.6 % and decreased by 3.2 %, respectively. In addition, the highest content of soluble protein was, respectively, 2.56 and 2.46 mg/g when the concentration of iodine in the form of iodide and iodate was at a dose of 600 mg/L. With respect to chlorophyll a content, the results of our work have shown that

N. flagelliforme

cells cultured with KI contained higher amounts of chlorophyll a than those treated with KIO

3

. All these studies suggest that the introduction of iodine in the form of KI proved to be much more effective in respect to iodine accumulation of

N. flagelliforme

than that of KIO

3

.

Honglei Fu, Yujie Dai, Yue Han, Lifang Yue, Feng Xia, Shiru Jia

Chapter 37. The Replacement of Phe28 by Ser Enhances the Stability of the GLP-1 Analog During Fermentation

Glucagon-like peptide-1 (GLP-1) is an incretin, which can effectively lower blood glucose levels, increase insulin secretion, and improve insulin sensitivity in patients with diabetes. Therefore, it is a potent endogenous insulin-stimulating hormone. Unfortunately, since GLP-1 can be degraded by the enzyme DPP-IV and NEP 24.11, its therapeutic benefit and fermentation yield are limited by the instability. Previous study has shown that replacement of the Lys34 with Arg could enhance the stability of GLP-1 in the plasma. In this study, to further improve the stability of the GLP-1, an additional mutation, replacement of Phe28 by Ser, was constructed, and then the protein was expressed in the recombinant

Pichia pastoris

, and analyzed by HPLC. The results show that this mutation could improve the stability of GLP-1 during fermentation.

Peng-Yan Li, Xue-Gang Luo, Qian Li, Wei Zhao, Hao Zhou, Tong-Cun Zhang

Chapter 38. Optimization of Fermentation Condition of Man-Made Bee-Bread by Response Surface Methodology

In research and development for drugs with thrombolytic function, natto kinase is commonly used for the treatment of thrombosis drug. The objective of this research is to use the Box–Behnken design to optimize the four fermentation parameters for activity of nattokinase to create bee-bread by a mixed culture with

Lactobacillus plantarum

MRS3,

Bacillus subtilis natto

ATCC15245, and fresh bee-pollen. The maximum yield of 801.29 IU/mL nattokinase activity was obtained after optimization experiments of culture conditions such as inoculum size 6.6 %, water size 35 %, fermentation temperature 33 °C, culture time 9 days (216 h), and pH stay at the natural range, and incubated the mixture in OMWS Oumai with a solid state cultivation, and finally obtained the new bee-bread products with high nattokinase activity and rich nutrient. The results demonstrated that exploiting functional food of bee-bread as both medicine and food is viable.

Chuanren Duan, Yongfen Feng, Hao Zhou, Xiaohua Xia, Yaning Shang, Yamin Cui

Chapter 39. Prediction of Lysine Acetylation Sites in Porcine Pancreas Lipase Modified by the Ionic Liquids Using Molecular Dynamics Simulations

Yi-Gang Jia, Yang Zhang, Hong-Man Zhang, He Huang, Lu-Jia Zhang, Yi Hu

Chapter 40. Effect of Oxygen on Fermentation Characteristics of Three Non-Saccharomyces from Hengshui Laobaigan

In this paper, effects of different oxygen concentrations on the fermentation characteristics of three non-

Saccharomyces

from Hengshui Laobaigan fermented grains were studied. Three non-

Saccharomyces

strains,

Pichia kudriavzevii

(Y3),

Pichia anomala

(Y4), and

Wickerhamomyces anomalus

, were isolated from fermenting grains in previous study. Because it is difficult to control oxygen concentration in solid state fermentation, the amount changes of fluid in containers were utilized to control oxygen. At the initial fermented stage (0 h), the volumes of liquid 50, 100, 150, 200, and 250 mL were corresponding to the oxygen concentrations 6.90, 6.71, 6.25, 4.32, and 4.50 mg/L, respectively. Three strains were separately incubated for 3 % yeast inoculation quantity and cultured in different amounts of sterilized medium for 6 days. The data shows that oxygen had been largely consumed in the stage of rapid yeast reproduction. Significance test concluded that different oxygen concentrations have significant effects on ethyl acetate. Reducing sugar was increased generally with increasing liquid volume; while alcohol yield of Y3 had no significant difference except for 50 mL, but Y4 and Y6 had remarkable differences in every liquid volume. 1-propanol was unaffected by liquid volume while different oxygen concentration has significant effect on isobutanol.

Huixia Zhu, Yuhang Zhang, Zexia Li, Yawei Guo, Zongzhi Cheng, Dongguang Xiao, Zhimin Zhang

Chapter 41. Microbial Transformation of Antitumor Isatin Derivatives by Fungi

The biotransformation of HKL-2c (

1

) and HKL-2h (

2

), the antitumor lead compounds found by our lab, were investigated. Compounds

1

and

2

were individually submitted to incubations with selected fungi

Trichoderma koningii

AS 3.4290,

Trichoderma viride

AS 2.2942,

Aspergillus flavus

AS 3.3950,

Aspergillus sydowii

AS 3.4258,

Aspergillus

AS 3.3885, and

Aspergillus niger

AS 3.3928. The products of hydrolysis from

1

and

2

by

Aspergillus

AS 3.3885 were identified based on spectroscopic methods. Those fungi, therefore, are useful for mild, selective hydrolysis of an ester of isatin substrates.

Xiaolin Peng, Kailin Han, Yan Wang, Peng Yu, Hua Sun

Chapter 42. Effect of Ultrasound on Lysine Muriate Crystallization

Ultrasound was applied to the crystallization process of lysine muriate solution. The effects on crystal yield of lysine muriate were investigated, including ultrasonic time, ultrasonic power, and crystallization time. The experimental results showed that the crystal yield reached 84.08 % with the adequate conditions of ultrasonic power 112 W, ultrasonic time 50 min, and crystallization time 5 h. And the effects of ultrasonic power on the nucleation induction period and crystal appearance of lysine muriate were also researched, the results indicated that with ultrasound, induction period was shortened significantly compared to that without it at the same supersaturation ratio. The higher the ultrasonic power, the shorter the induction period, and the smaller the crystals. With the increase of ultrasonic power, the crystals became smaller and more uniform. It can be concluded that using ultrasound-assisted crystallization cannot only improve the crystallization rate and crystal yield, but also obtain crystalline product with the characteristics of small particles and uniform distribution by controlling ultrasonic power.

Aijun Hu, Zili Chen, Shuting Jiao, Huanqin Peng, Yanshu Fan, Lin Chen, Meiling Liu, Jie Zheng

Chapter 43. Isolation and Identification of a Bacillus amyloliquefaciens Strain Against Grape Downy Mildew and Optimization for Its Liquid Fermentation Medium

An antagonistic bacterial strain named J12 against the grape downy mildew was isolated from 92 bacteria on grape leaves. Antagonistic strains were isolated by exploiting streak plate method. J12 was identified based on morphology observation, physiological and antibacterial characterizations, 16S rDNA sequence analysis, and the phylogenetic tree. The controlling effect of J12 on grape downy mildew was 78.6 %. The liquid fermentation medium of J12 was optimized with the response surface methodology to improve the number of live bacteria. The results showed that the strain J12 was classified as

Bacillus amyloliquefaciens

. For its liquid fermentation medium, the optimum carbon source was sucrose, the best nitrogen source was soybean meal, and the best C/N ratio was 3:1. By Plackett–Burman design and response surface methodology, the best culture medium formula is 31.18 g/L sucrose, 10.57 g/L soybean meal, 2.206 g/L K

2

HPO

4

, 2.5 g/L yeast, 0.5 g/L MgSO

4

·7H

2

O. Theoretic optimum cell number was 90.87 × 10

8

cfu/mL. The number of bacteria in three parallel experiments was 92.54 × 10

8

cfu/mL, indicating that the equation fits well to the actual production.

Jiping Guo, Guang Ma, Huixia Zhu, Junfan Fu

Chapter 44. The Adsorption Properties of Macroporous Resin for Fusel Oil of Luzhou—Flavor Liquor

The D3520 resin was suited for adsorption and desorption of fusel oil by the comparison study of different types of resin. The study confirmed that macroporous resin D3520 had favorable adsorption and desorption capability by comparing different resins based on adsorption quantity, adsorption rate, and desorption rate. Temperature affected the fusel oil adsorption rate of D3520 and with more impact the temperature was high. There was little difference in the adsorption rate at 25 and 30 °C and the rate was up to 58.85 %. Among all strippant, the effect of acetone was higher than all concentration ethyl alcohol, up to 65.80 %. After treatment by D3520, there was little difference in physical, chemical, and sensory indexes of Luzhou flavor liquor and was not a bad influence on Luzhou flavor liquor. The Luzhou flavor liquor was soft and well balanced, with more rich ester aroma, and the wine quality was better by the D3520 resin treatment.

Jinxu Sun

Chapter 45. Low Labeling 13C Metabolic Flux Analysis of Saccharomyces cerevisiae Using Gas Chromatography–Combustion–Isotope Ratio Mass Spectrometry

The applicability of gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) for the quantification of

13

C enrichment of proteinogenic amino acids in metabolic tracer experiments was evaluated in this paper. We measured the

13

C enrichment of proteinogenic amino acids in hydrolyzates using GC–C–IRMS from a series of parallel batch cultivations of

Saccharomyces cerevisiae

, which was cultured by mixtures of natural glucose and [1−

13

C] glucose, containing 0, 0.5, 1, and 2 % [1−

13

C] glucose, respectively. By decreasing the [1−

13

C] glucose content, kinetic isotope effects played an increasing role but could be corrected. The

13

C metabolic algorithm and matrix algorithms were optimized in this study. The central metabolism of vivo fluxes were determined by the calculation method optimization. The obtained flux distribution was similar to published results, which obtained from GC–MS method using conventional high labeling (99 %). The GC–C–IRMS-based method involves low labeling (0.5 %) degree of expensive tracer substrate, and suits well for larger laboratory and industrial pilot-scale fermentations.

Qi-ding Zhong, Guo-hui Li, Dong-dong Zhao, Dao-bing Wang, Shi-gang Shen, Zheng-he Xiong

Chapter 46. A Comparative Study on the Antioxidant Activity of Two Polysaccharides from Ganoderma lucidum

Two polysaccharides from

Ganoderma lucidum

(GLP1 from fermentation broth and GLP2 from fruiting body) were obtained and investigated for their in vitro antioxidant properties. The two polysaccharides showed strong antioxidant activities in a dose-dependent manner. At 5.0 mg/mL, the reducing power and the scavenging rate on DPPH radical and hydroxyl radical of GLP1 were 1.25, 50 and 80 %, while that of GLP2 were 1.46, 85, and 78 %, respectively. GLP1 and GLP2 also showed strong protective effects on yeast cells from UV and H

2

O

2

damage. At 20 mg/mL, the protective effects on yeast cells from UV and H

2

O

2

of GLP1 were 57 and 49 %, and that of GLP2 were 84 and 79 %, respectively. Thus, both GLP1 and GLP2 could be considered as antioxidants for food and pharmaceutical industries.

Ruyu Tao, Limin Hao, Shiru Jia, Xin Zheng, Jianyong Yu, Qingwu Jiang

Chapter 47. Biofortification Using Bacteria Containing an Atrazine-Degrading Gene and Its Effects on Reactor Operating Efficiency

To investigate the effects of biofortification of activated sludge reactors using genetically engineered microbes containing a plasmid encoding atrazine chlorohydrolase, we compared biodegradation efficiency and sludge properties during three operating stages in a conventional activated sludge (CAS) reactor and a MBR. Our results show that with the addition of genetically engineered bacteria and the selection for indigenous biodegrading microbes, membrane fouling was reduced. Atrazine has a certain level of biotoxicity toward activated sludge and shows an inhibitory effect toward pollutant removal. After biofortification, atrazine degradation was superior in MBR than that in CAS reactors. The atrazine removal rate was 92.6 % in the MBR, while it was 82.6 % in the CAS reactor. Following biofortification, the sludge concentration in the MBR was maintained at 7.3 g/L, while the sludge concentration in the CAS reactor was 2.3 g/L.

Yue Wang, Jinxu Sun

Chapter 48. A Novel One-Pot Five-Component Synthesis of Tetrahydro-pyrrolo[3,4-b]pyridine-5-one via Ugi/Aza-Diels–Alder Tandem Reaction

A novel five-component domino processes to tetrahydro-pyrrolo[3,4-b]pyridine-5-one (

8

) were developed. Reaction of 5-phenyloxazole-2-carbaldehyde (

1

),

p

-toluidine (

2

), (Z)-4-methoxy-4-oxobut-2-enoic acid (

3

), and

t

-butyl isocyanide (

4

) in methanol at 60 °C provides an efficient access to the drug-like bicycle compound (

8

) in a good yield. In this one-pot process, a traditional oxo-bridged tricycle intermediate was formed via Ugi/ara-Diels–Alder tandem reaction and then the solvent, methanol, sequentially attacks and cuts off the oxo bridge under mild conditions via SN2 reaction. Formation of one C–N, two C–O, and three C–C bond with the triple domino sequence (Ugi/IMDA/SN2) is involved in this new scaffold generating reaction. The operational simplicity and maximizing the buildup of structural complexity makes this novel multicomponent reaction valuable in diversity-oriented synthesis of tetrahydro-pyrrolo[3,4-b]pyridine-5-one.

Yan Liu, Tianyi Shang, Chuanming Xu, Hui Yang, Peng Yu, Kui Lu

Chapter 49. Study on Preparation and Application of High Esterification Green-Liquor Daqu

The optimal conditions of esterification Daqu production requires medium temperature to be used in the Daqu making process. The ratio of raw materials is 6:3:1 (wheat: barley: pea), the inoculation of

Monascus

(

Monascus purpureus

) FBKL3.0018 is 10 g/kg, and the water of Daqu is controlled at 40 %. The highest temperature is more suitable, which is controlled at 55 °C, and the culture time is 25–28 days. The best storage period of Daqu esterification is 2–3 months. This study could improve the quality of Daqu, the development, and research of fortified Daqu, which will become a hot point in increasing the quality rate of Luzhou-flavor liquor.

Xiao-Dan Wang, Bei Xiao, Shi-Dong Ban, Si-Xia Xu, Xi-Cui Shen, Shu-Yi Qiu

Chapter 50. Metabolomics Analysis Between Wild-Type and Industrial Strains of Streptomyces avermitilis Based on Gas Chromatography–Mass Spectrometry Strategy

Objective

To conduct a metabolic analysis of the intracellular metabolites in

Streptomyces avermitilis,

so as to search for possible biomarkers and to discover the mechanism of higher avermectin production.

Methods

GC–MS analysis was used to obtain the fingerprint of wild-type and industrial

S. avermitilis

9-39 strains.

Results and conclusion

multivariate statistical analysis demonstrated that

d

-cellobiose,

d

-galactose,

d

-glucopyranose,

d

-mannose,

d

-turanose, glutamine,

l

-serine, and maltose were mainly responsible for distinguishing the wild-type

S. avermitilis

and industrial strain 9-39, and most of them belong to glycometabolism. Thus, strengthened glycometabolism is the reason for high yield of avermectin in

S. avermitilis

.

Gang Guo, Ping-ping Tian, Dan Tang, Xiaoxia Wang, Hong-jin Yang, Peng Cao, Qiang Gao

Chapter 51. Application of Orthogonal Design to Optimize Fermentation Conditions of Bacillus amyloliquefaciens BI2

Bacillus

amyloliquefaciens

BI

2

, isolated from corn straw silage, exhibits to be antagonistic against many kinds of molds, especially

Aspergillus flavus

. In this work, optimization of the medium and culture conditions was studied by adopting the combining single-factor tests and an orthogonal experiment. On the study of the medium optimization, yeast powder, monosodium glutamate, sodium nitrate, and the proportion of starch and maltose were selected as four factors at three levels each. On the study of the optimizing culture conditions, medium volume, culture temperature, fermentation time, and inoculums size were used as four factors at three levels each. The results demonstrated that yeast powder and monosodium glutamate have significant effects on medium optimization; medium volume, culture temperature, and fermentation time give crucial influences on culture conditions, and the influences of the other factors are not remarkable. The optimized medium results shown by the interaction tests were as following: yeast powder 1.5 %, monosodium glutamate 2 %, sodium nitrate 0.8 %, starch 0.17 %, and maltose 0.33 %; the optimal culture conditions were displayed as following: medium volume 60 mL, culture temperature 30 °C, inoculums size 2 %, and fermentation time 36 h.

Yajun Wang, Zhanglei Cao, Miao Yu, Depei Wang

Chapter 52. Optimization of Fermentation Medium for Citric Acid Production by Aspergillus niger

A statistically based method was used to optimize the fermentation medium for enhancing citric acid production by

Aspergillus niger

TCCC 41661. The optimization of single-factor experiments (soy peptone, (NH

4

)

2

SO

4

, dextrin) and an orthogonal experiment were applied to determine the optimal concentration of each significant variable. The optimum values for the critical components were obtained as follows: 0.25 g/L soy peptone, 12.0 g/L dextrin, 2.0 g/L KH

2

PO

4

, 2.0 g/L (NH

4

)SO

4

, 0.1 % Tween 80. Under this condition, the citric acid production of 16.11 g/L was obtained.

Jianhua Zhang, Kun Li, Juan Huang, Depei Wang

Chapter 53. Study on the Soxhlet’s Extraction of Star Anise Oil and Preliminary Investigation of Its Antibacterial Activity

In this study, Soxhlet extraction method using ethyl acetate as a solvent was used to extract the volatile oil from star anise fruits. The best technological extraction conditions were obtained by a series of single-factor experiments. The antibacterial activity of star anise oil obtained by Soxhlet extraction method was preliminary determined. The result showed that the most important factor affecting the extraction rate of oil from star anise fruits was extraction temperature, followed by powder granularity, solid–liquid ratio, and extraction time. The best extraction conditions were as follows: 1:22 (w/v), 40 min, 120 °C, and 60–80 mesh, respectively. Under these conditions, the yield of star anise oil reached 25.9827 %. It was proved that star anise oil extracted by Soxhlet extraction method could reduce the cost of investment equipments, improve production significantly, and keep the high-quality and natural flavor of the essential oil. Antibacterial experiment demonstrates that star anise oil method has a strong inhibitory effect against

Escherichia coli

and

Bacillus subtilis

, so that it could be applied as a natural and environment-friendly antibacterial agent in many areas, such as food products and pharmaceutical sectors.

Lin Tian, Ping Li

Biological Separation and Biological Purification

Frontmatter

Chapter 54. Production of Diosgenin from Dioscorea zingiberensis by Mixed Culture of Three Filamentous Fungi

The mixed culture of

Aspergillus oryzae

,

Phanerochaete chrysosporium,

and

Aspergillus niger

was explored for the production of diosgenin from

Dioscorea zingiberensis

. Conditions for biotransformation of saponin using mixed culture of three fungi were optimized. The maximum production of diosgenin (40.06 ± 0.53 mg/g) was obtained with

D. zingiberensis

concentration (50.00 g/L), inoculation size (2.0 mL of spore suspension, 1:2:3 ratio of

A. oryzae, P. chrysosporium

, and

A. niger

, v/v/v), initial pH (5.0), and cultivation time (6 days). In the optimization conditions, mono and mixed cultures were tested, with mixed culture of three fungi giving better yields of diosgenin and β-glucosidase. The diosgenin yield of optimization biotransformation increased by 50.54 % compared to the traditional acid hydrolysis method (26.61 ± 0.78 mg/g). The use of mixed culture of these fungi proposes an approach for more efficient and clean diosgenin production by

D. zingiberensis

.

Hua Xiao, Linlin Huang, Jinxia Xie, Songtao Bie, Yu Li

Chapter 55. Characterization of Volatile Constituents of Chinese Hawthorn (Crataegus spp.) Fruit Juices

All hawthorn fruits used in this study were collected from four provinces of the China namely Shandong, Shanxi, Hebei, and Liaoning. Hawthorn fruits were prepared for hawthorn fruit juices. Volatile compounds in the juices of 12 cultivars of hawthorn were determined by gas chromatography and mass spectrometry (GC–MS) coupled with headspace solid-phase micro-extraction (HS–SPME) method. Semi-quantitative analysis was employed for the analysis of volatile constituents in hawthorn juice. Among 61 volatile compounds detected in the 12 samples, 21 volatiles were found in all samples. Esters and alcohols were the main volatiles present in the hawthorn fruit. Aldehydes, aromatic compounds, furans, terpenes, acids, and sulfurs also made great contributions to the total volatiles of the hawthorn fruits. Ethyl acetate, 3-methylbutanol, 3-octanone, acetic acid, furfural, dimethyl sulfide, and

p

-menth-1-en-8-ol were suggested to the major volatile compounds in all samples. For most volatile compounds identified in the current research, their content in sample 7 was highest than the others and the sample with lowest contents was sample 6.

Yuping Zhao, Yangyang Wang, Jiwu Wang, Zhilian Wu, Zuli Sun, Tiantian Tian, Hao Niu, Lili Jing, Zhengyu Fang, Jianrong Yang

Chapter 56. Optimization of Extraction Conditions for Crude Antibacterial Proteins/Peptides from Clarias gariepinus By-products

In this paper, acetic acid was used for extracting crude antimicrobial proteins/peptides from by-products of healthy

Clarias gariepinus

reared at high stocking density. For the extraction process, three variables, namely the concentration of acetic acid, extraction time, and the ratio of liquid to solid were optimized by using orthogonal design L

9

(3

3

) with the yield and antibacterial activity of the crude proteins/peptides extracts as test indices. The yields of extracted crude proteins were analyzed by range analysis. The results showed that the yield of crude proteins/peptides was different among nine groups, the highest was group 7, producing 1.5842 g (dialyzed in 1,000 MWCO and lyophilized) per 100 g raw material. All the nine extracted samples had similar inhibitory activity against

Aeromonas hydrophila,

Escherichia coli

,

Staphylococcus aureus,

Citrobacter

sp., and

Microbacterium

sp. except that groups 7 and 9 exhibited higher inhibitory activity against

A. hydrophila

and

E. coli

; moreover, the inhibitory activity of group 7 was higher than group 9 against the two bacteria strains. Nine groups did not display obvious different patterns on the SDS-PAGE gel. Based on these results above, the final conclusion was that group 7, namely 10 % acetic acid, 12 h extraction, and 5:1 liquid to solid ratio was the best combination for isolating the crude antimicrobial proteins/peptides from by-products of

C. gariepinus

.

Yan Wang, Yunxia Xu, Junyan Mei, Chengxun Chen, Xiaomei Wang

Chapter 57. Screening, Isolation, and Identification of Bacillus coagulans C2 in Pu’er Tea

Pu’er Tea is a popular post-fermented tea and has a variety of health benefits. Many microorganisms, especially lactobacillus, exist and play an important role in the fermentation process of Pu’er tea. In this study, we try to screen some lactobacillus by plate method from the Pu’er tea, and strain C2 with significant cycle isolated from Pu’er tea. 16S rRNA gene sequencing results showed that strain C2 had 99 % sequence similarity to that of the type strain of three

Bacillus coagulans

(NR 102791.1, CP002472.1, NR 115727.1), and the results show that methyl red test and V&P test were positive. Sugar fermentation test suggested that strain C2 produced acid but not gas, which is identical to

B. coagulans

. This study confirmed that lactic acid bacteria play a role in Pu’er tea fermentation.

Cuixia Feng, Zhongyuan Li, Kun Li, Minghui Zhang, Cuiqiong Wang, Xuegang Luo, Tongcun Zhang

Chapter 58. Production and Identification of Antifungal Compounds Produced by Bacillus subtilis B579

Biological control has become an important approach to suppress many pathogens.

Bacillus subtilis

is considered to be an excellent biocontrol agent not only due to its ability on inducing plant systematic resistance, but also on producing various hydrolytic enzymes and antibiotics. In this study, polymerase chain reaction (PCR) was used to detect the 12 genes related to the antifungal compounds biosynthesis. Six genes were detected that exist in the genome DNA of B579 by the sequence homology analysis. Five genes were related to biosynthesis of lipopeptide antifungal compounds, and one gene was related to biosynthesis of protein antifungal compounds. Lipopeptide antifungal compounds were obtained from the supernatant of B579 using the method of acid deposition and methanol extraction. Two homologies with the molecular weight of

m/z

1043.59 and

m/z

1057.35 were detected in the lipopeptide antifungal compounds, which had the similar molecular weight with iturin A. Three homologies with the molecular weight of

m/z

1008.32,

m/z

1022.06, and

m/z

1036.13 were detected in the lipopeptide antifungal compounds, which had the similar molecular weight with surfactin. The antifungal compounds produced by B579 could be of a good prospect for being used as a new tool for biological control.

Fang Chen, Yu Zheng, Jianmei Luo, Deduo Han, Min Wang

Chapter 59. Application of Molecularly Imprinted Polymers in Purification and Separation for Epothilones

A highly effective purification process combined with molecularly imprinted polymers (MIPs) was developed to selectively isolate Epothilones (Epos) from a complex milieu. The MIPs of Epos were successfully synthesized by precipitation polymerization, and the optimal ratio of the template molecule to functional monomer and cross-linker was 1:4:20 (with the molar ratio). Under the optimized condition, the resulted products were characterized by scanning electron microscope (SEM). The binding properties of the MIPs were evaluated by the adsorption kinetics and static adsorption. From the correlation coefficients (

R

2

) of the fitting models, the equilibrium data fitted well to Freundlich model (

R

2

= 0.9987), indicating multilayer adsorption. Finally, we successfully applied the MIPs in the solid-phase extraction (SPE) process of Epos, and its purity was improved more than 40 % than the origin. It achieved an effectively selective Epos cleanup procedure, sequentially established a favorable foundation for later research on Epos.

Ruicheng Sun, Lin Zhao, Jikun Yang, Naiqiang Wang, Xinli Liu

Chapter 60. Identification of Peanut Intersectional Hybrids with SSR Markers

Wild peanut relatives constitute an invaluable gene source for broadening the narrow gene base of the cultivated peanut. However, severe pre-/post- fertilization obstacles impeded the utilization of incompatible species within the

Arachis

genus. In the present study, through SSR profiling, three true intersectional F

1

hybrids of Qunyu 101 ×

Arachis pusilla

and a true intersectional F

1

hybrid of Huayu 40 ×

A. pusilla

resulting from post-pollination hormone treatment of flower bases were identified, providing strong evidence for the effectiveness of the in vivo hormone treatment method in overcoming cross-incompatibility in the genus

Arachis

.

Shuo Meng, Xiu Zhen Wang, Yue Yi Tang, Qi Wu, Quan Xi Sun, Chen Jiang, Chuan Tang Wang, Li Feng Liu

Chapter 61. Partial Purification and Chemical Characterization of a Bioemulsifier and Its Application in MEOR

The bioemulsifier secreted by strain XS2, which was isolated from oil-contaminated soil samples in Yumen oil field, China, was partially purified and analyzed. Protease hydrolysis and heat treatment results showed that the protein composition of the bioemulsifier played a significant role in emulsification activity. High-performance liquid chromatography (HPLC) study suggested that the protein composition may exist as glycoprotein. Monosaccharide analysis by gas chromatograph–mass spectrometer (GC–MS) showed that galactose, glucose, xylose, and mannose presented in the bioemulsifier. The emulsification activity of this bioemulsifier was relatively high due to different carbohydrates (

n

-hexadecane 61 %, liquid paraffin 60 %, Toluene 77 %). Furthermore, single well stimulation trial in Changqing oil field, was done to explore the potential application in Microbial-enhanced oil recovery (MEOR). Up to September 2014, the cumulative increased crude oil production was about 88 T in total 240 days, and the water content of the well-produced liquid decreased from 85 to 25 %. These data illustrated that the bioemulsifier performed a high potential in applications and had important economic values.

Dayuan Dong, Xingbiao Wang, Mingyu Cai, Jingjing Wang, Yifan Han, Xiaoxia Zhang, Zhiyong Huang

Chapter 62. Optimization of a Whey Containing Medium for β-Galactosidase Production by Lactobacillus reuteri

Lactobacillus reuteri

MF2-2 was screened out for producing a high level of β-galactosidase from chicken fecal samples and identified with 16S rRNA gene sequence analysis. To reduce production cost, a new whey containing medium was formulated on the basis of MRS medium and optimized. Nutrients including whey, yeast extract, NH

4

H

2

PO

4

, and vitamin B

1

were investigated at the individual and interactive level by the Taguchi orthogonal tests. The production of β-galactosidase was improved from 490.0 U/g in lactose-based MRS (L-MRS) medium to 814.6 U/g in the optimized whey-based MRS (W-MRS) medium. W-MRS medium did not comprise of lactose, peptone, and beef extract and was expected to cut the raw material cost approximately in half compared to L-MRS medium.

Mengfei Li, Huiming Zhu, Huibin Qin, Yan Zhang, Hongjiang Yang

Chapter 63. Enzymatic Bioconversion for γ-Aminobutyric Acid by Lactobacillus brevis CGMCC No. 3414 Resting Cells

In this work, γ-aminobutyric acid (GABA) was prepared by the decarboxylation of

l

-glutamate via

l

-glutamate decarboxylase in the resting cells of

Lactobacillus brevis

CGMCC No. 3414. The influence of cell concentration, cell age, buffer system, reaction time, and substrate concentration were investigated. The optimal composition of bioconversion system was composed of 50 g/L resting cells, cell age at 48 h fermentation, 0.2 M disodium hydrogen phosphate–citric acid buffer, and 25 mM monosodium glutamate. When the bioconversion system was performed at pH 4.6, 30°C, and 180 r/min shaking for 4 h, GABA production in biotransformation solution was 23.29 mM and the molar yield rate of bioconversion reached 93.15 %.

Xiu-feng Shi, Bo Zheng, Chuan-you Chang, Peng Cao, Hong-jin Yang, Qiang Gao

Progress of Biotechnology

Frontmatter

Chapter 64. [FeFe]-Hydrogenase: Catalytic Center and Modification by Genetic Engineering

In this review, we highlight the [FeFe]-hydrogenase, which is capable of catalyzing the splitting of molecular hydrogen to produce electrons and protons or catalyzing the reversible reaction: 2H

+

+ 2e

= H

2

↑ as a potential renewable fuel. We have focused on [FeFe]-hydrogenase because of structural studies have shed more light on the hydrogenase activity than the [NiFe]-hydrogenase. Our studies on the [FeFe]-hydrogenase from

Chlamydomonas reinhardtii

CC-503 (

HydA

1

) have also been highlighted. There are two factors influencing the multiplexed hydrogenase activity: a single hydrophobic channel of catalytic center, which is known as the H-cluster active site based on site-directed mutagenesis, moreover, some promising results have already been obtained. Modifications of [FeFe]-hydrogenase can improve its stabilization and activity in vitro, increase the efficiency of bioenergy utilization, and promote industrial amplification of biofuel production.

Jiayi He, Chunfei Wu

Chapter 65. Characteristics of Staphylococcus aureus Isolates from Raw Milk

Staphylococcus aureus

has raised great health concerns worldwide. In this work, with the traditional culture-based methods, totally 70 bacterial isolates on Barid–Parker medium from raw milk samples are collected from 181 healthy cows in a dairy farm located in Northern China. With comparative sequence analysis of 16S rDNA genes, 18 isolates were identified as

S. aureus

. One isolate was resistant to cefoxitin and tentatively considered as a methicillin-resistant

S. aureus

(MRSA) strain.

Spa

typing was performed to discriminate the strains. Four

spa

types were determined, including t3104 in 12 strains, t267 in 3 strains, t521 in 2 strains, and t528 in strain Z1584. The type t11557 was a new

spa

type discovered in this work and its repeats succession was 07-12-21-13-34-34-33-13. Our work suggested that the prevalence of MARA strains was quite low in fresh raw milk produced in the dairy farm located in Northern China.

Xiaomei Zhang, Qian Li, Hongjiang Yang

Chapter 66. EST-SSR Marker-Based Assay for Purity Identification of Melon “Green Angle”

In order to define the purity of melon (

Cucumis melo

L.) “Green Angle” variety F1 seed, PCR amplification, detection, and analysis were carried out with leaf DNA, using EST-SSR molecular marker technology. The results show that, among 57 SSR primers, PCR products with one pair of primer showed polymorphism from parents. For this marker, parents showed single allele whereas the hybrids showed both the parental alleles indicating the heterozygosity of the hybrids. 51 seeds were tested using the marker, and one seed was off-type. The same lot of seeds was also tested in the field and the purity was 98.4 %. It showed that the EST-SSR marker-based PCR assay is effective in determining the purity of F1-hybrid seeds of melon “Green Angle”.

Ou-Jing Li, Xiao-Mu Chen, Pu-Xian Xia, Zhong-You Pei, Yong Wang, Qing-Kuo Lan, Ruo-Wei Zhang

Chapter 67. Exposure to Static Magnetic Fields Affects Insulin Secretion in INS Cells

The exposure to static magnetic fields (SMFs) has rapidly increased and is recently accompanied with the wide use of more and more electrical appliances. Although few researches have been reported on other types of magnetic fields that may have the effects on clinical therapy of diseases, it is rarely seen reports on the effects of moderate static magnetic fields on human health. In this study, in order to evaluate the influence of exposure to moderate static magnetic fields on biological systems, we cultured the INS-1 under exposure to moderate intensity of 400 mT and sham conditions for different times from 6 to 18 h, insulin secretion to medium and mRNA expression will be tested after exposure. Insulin secretion has significantly increased after exposed to SMFs with moderate intensity of 400 mT for 18 h compared with the sham condition. Furthermore, similar to the insulin secretion, insulin 1 and insulin 2 mRNA expression also increased after exposure. These results suggest that exposure to SMFs at 400 mT position for specific times might have positive stimulation in both insulin secretion and insulin mRNA expression, and these will be used hopefully in further investigations in vitro, even in the therapy clinically.

Libin Mao, Zhixia Guo, Huiqin Wang, Qiongyao Wu, Nan Wang, Tong-Cun Zhang

Chapter 68. The New Strategy of Breeding Cytidine Excessive Biosynthesis Mutants by pyr Operon Rearrangement of Bacillus amyloliquefaciens

Cytidine is a good antitumor and antiviral intermediate which can also be used as a healthy food ingredients. With the market for cytidine increasing, the large-scale production of cytidine by microbial fermentation method has become a major way to solve this problem. While improving cytidine production, the regulation of pyr operon is essential to the excessive synthesis cytidine. However, its transcriptional regulation mechanism is unclear. The passage summarizes the regulation of de novo pyrimidine nucleotide biosynthesis (pyr genes) and the metabolic regulation mechanism of cytidine synthesis in

Bacillus amyloliquefaciens

. This paper makes the regulation protein pyrR as the research object, presents a series of

Bacillus amyloliquefaciens

pyrimidine operon transcriptional regulatory rearrangement strategy, effects the transcriptional regulation on cytidine biosynthesis, and aims to provide a theoretical basis for cytidine yielding strain.

Qing Wu, Huiyan Liu, Haitian Fang, Jianguo He, Xiaoguang He, Linan Yu
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