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2015 | Book

Advances in Applied Biotechnology

Proceedings of the 2nd International Conference on Applied Biotechnology (ICAB 2014)-Volume I

Editors: Tong-Cun Zhang, Motowo Nakajima

Publisher: Springer Berlin Heidelberg

Book Series : Lecture Notes in Electrical Engineering

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About this book

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.

Table of Contents

Frontmatter
Erratum to: Characterization of Recombinant L-Amino Acid Deaminase of Proteus mirabilis
Chenglin Zhang, Jia Feng, Xixian Xie, Qingyang Xu, Ning Chen

Microbial Genetics and Breeding

Frontmatter
Chapter 1. Cloning and Bioinformatics Analysis of spsC Gene from Sphingomonas sanxanigenens NX02

Sphingomonas sanxanigenens

strain NX02 synthesizes a novel sphingan Ss, which can be used as drilling mud and thickening agent in the recovery of petroleum by water flooding. In order to research genes involved in the biosyntheses of sphingan Ss, strain NX02 was induced by transposon mini-Tn5 on suicide plasmid pUT, and a mutant strain T163, which cannot produce sphingan Ss, was screened. The

spsC

gene of NX02 was obtained by the method of Tn5 flanking PCR and LP-RAPD. The predicted amino acid sequence of the

spsC

protein possessed 493 amino acids and a calculated molecular mass of 53.5 kDa. Bioinformatics analysis revealed that

spsC

had the highest 60 % amino acid sequence identity with polysaccharide biosynthesis protein of

Novosphingobium lindaniclasticum

LE124.

spsC

protein had typical polysaccharide polymerases family transmembrane helices, located between amino acids Y13-V44 and P411-L436. The N-terminal sequence of

spsC

had high identity to chain length determinant protein of Wzz superfamily.

Xiaoyan Li, Haidong Huang, Mingming Zhou, Peng Zhang
Chapter 2. Preliminary Study on Salt Resistance Seedling Trait in Maize by SRAP Molecular Markers

In this study, different genotypes of maize salt tolerance inbred line and salt sensitive inbred line were used as the parent hybrid combinations to obtain F2 populations. Two salt tolerance extreme types of DNA pools were established, where BSA method was used to select polymorphic SRAP markers. The result showed that 48 pair primers can be amplified and clear and stable bands can be obtained by parental, tolerant, and sensitive gene pools. Six pair primers of M2E1, M2E7, M6E15, M7E7, M11E4, and M14E6 showed polymorphism between two parents and between tolerant and sensitive bulks. The six SRAP molecular markers closely linked to salt tolerance were determined. The best maize SRAP-PCR reaction system was established. This research will accelerate maize marker-assisted selection breeding and lay the foundation for salt-tolerant gene cloning.

Chunyang Xiang, Jin Du, Peipei Zhang, Gaoyi Cao, Dan Wang
Chapter 3. Isolation of Differentially Expressed Genes from Groundnut Genotypes Differing in Seed Dormancy

In situ sprouting may result in considerable reduction in quantity and quality of groundnut produce. The most feasible and convenient solution is to develop groundnut cultivars with seed dormancy. No molecular marker or gene associated with this important trait of groundnut has been documented. In the present study, 105 unique cDNA fragments were isolated from two groundnut materials with contrasting dormancy phenotypes using GeneFishing technology. Three of the 16 DEGs were related to seed germination as suggested by BLAST2GO analysis. Differential expression of a gene with functions indicated in seed germination [85-3-1 (oleosin kda-like)] was confirmed by qRT-PCR. We postulate that expression of the gene is positively related to intensity of groundnut seed dormancy, and that the gene product protects oil bodies thereby preventing lipid mobilization from providing energy for germination.

Bo Qu, Yue Yi Tang, Xiu Zhen Wang, Qi Wu, Quan Xi Sun, Shu Yan Guan, Chuan Tang Wang, Pi Wu Wang
Chapter 4. Increase of the Lycopene Production in the Recombinant Strains of Escherichia coli by Supplementing with Fructose

Lycopene is an important carotenoid which has been widely used as functional foods and feed supplements, and pharmaceutical compounds. In the present study, the host strain and the auxiliary carbon source were optimized to enhance the lycopene yield in the recombinant

Escherichia coli

. The results showed that lycopene concentration of the cells could be significantly increased when the recombinant strain was grown on the LB medium supplemented with 6 g l

−1

fructose, whereas glucose has little effect. Under the optimized conditions, the recombinant bacteria could exhibited a lycopene yield up to 1,050 mg l

−1

for 24 h in a 250 ml baffled flask. This is the first report of enhancing lycopene production by supplementing with fructose.

Tong-Cun Zhang, Wen Li, Xue-Gang Luo, Cui-Xia Feng, Ming-Hui Zhang, Wen Du, De-Yun Ma
Chapter 5. Isolation of Differentially Expressed Genes from Developing Seeds of a High-Protein Peanut Mutant and Its Wild Type Using GenefishingTM Technology

Peanut is a good source of dietary protein. Raising protein content in peanut will not only fill the growing need for vegetable protein, but also in most cases lower oil content, which is good news to health-conscious populations. However, no attempt has been made to isolate genes related to protein content in peanut. In the present study, a total of 40 unique differentially expressed genes in developing seeds of high-protein peanut mutant (SDPM) and its normal-protein wild type (SDPW) at 46 or 49 days after flowering were isolated using Genefishing technology. Of them, 8 sequences were undescribed previously; the rest 32 were found to be significantly similar to the sequences in GenBank nr database. Three genes potentially related to protein content in peanut, viz., P2-2-2, P2-92-2 and P1-89-1-5 with high homology to thioredoxin h, arachin ahy-4 and abc transporter, respectively, were selected for further analysis. All the 3 genes validated by qRT-PCR showed differential expression between SDPM and SDPW, with relative expression ranging from 0.41–10.60. The detailed functions of the differentially expressed genes isolated from developing seeds in the present study in conditioning peanut seed protein content are yet to be validated by transgenic experiments.

Shu Tao Yu, Hong Bo Yu, Guo Qing Yu, Li Ren Zhao, Hong Xi Sun, Yue Yi Tang, Xiu Zhen Wang, Qi Wu, Quan Xi Sun, Chuan Tang Wang
Chapter 6. Identification of the Binding Domains of Nedd4 E3 Ubiquitin Ligase with Its Substrate Protein TMEPAI

To investigate which domains of Nedd4 are responsible for the interaction with its substrate protein TMEPAI, the plasmids encoding GST-tagged WW domains of Nedd4 and His-HA-tagged TMEPAI were constructed, and the fusion proteins were expressed in

E. coli

BL21-CodonPlus (DE3)-RIL cells and purified. GST pull-down experiment indicated that Nedd4 directly interacts with His-HA-TMEPAI, and the WW2 and WW3 domains of Nedd4 are responsible for the interaction with TMEPAI, which represents a general mechanism for the interaction between Nedd4 and its substrate protein.

Lei Jing, Xin Huo, Yufeng Li, Yuyin Li, Aipo Diao
Chapter 7. Optimization of the Fermentation Conditions of Pep-1-Fused EGF in Escherichia coli

Human epidermal growth factor (hEGF), a well-known polypeptide agent which has been widely used in the medicine and cosmetics, is a 6.2 kDa single-chain polypeptide consisting of 53 amino acids. Here, to enhance its transmembrane ability, a recombinant EGF fused with Pep-1, a cell-penetrating peptides (CPP) that has been previously shown to be powerful transport vector tool for the intracellular delivery of a variety of cargos through the cell membrane, was expressed in

Escherichia coli

. Furthermore, The expression conditions was optimized, and the results showed that the Pep-1-fused EGF (P-EGF) could be successfully expressed in

E. coli

BL21-TrixB (DE3) using an expression vector, pGEX-6P-3, which contains a GST tag. The recombinant product reached the highest soluble expression when the expression strain was induced by 0.2 mmol/l IPTG and cultivated at the temperature of 20 °C with a rotation speed of 200 rpm for 8 h.

Tong-Cun Zhang, De-Yun Ma, Xue-Gang Luo, Yue Wang
Chapter 8. Characterization of Rhamnolipid Production in a Pseudomonas aeruginosa Strain

Rhamnolipid is a class of glycolipid biosurfactant and has the potential to replace chemical surfactants due to its low toxicity and biodegradability. In this study, 38

Pseudomonas

strains were screened for rhamnolipid production on cetyl trimethyl ammonium bromide (CTAB)-methylene blue agar plates. Among them, strain 487 was found to produce the relative high level of rhamnolipid. The rhamnolipid extract could reduce the surface tension of the medium from 67.2 to 32.7 mN/m and form stable emulsions with liquid paraffin. Strain 487 was identified as

Pseudomonas aeruginosa

with 16S rDNA gene sequence analysis. Rapeseed oil and glycerol were investigated for rhamnolipid production. Strain 487 produced 42.4 g/L rhamnolipid in rapeseed oil medium versus 33.4 g/L rhamnolipid using glycerol as carbon resource after 72 h fermentation. In addition, thin layer chromatography (TLC) and Fourier transform infrared spectroscopy (FTIR) were performed to analyze the structure of the rhamnolipid molecule synthesized by strain 487.

Cuikun Zhang, Hongjiang Yang
Chapter 9. High-Quality Protein-Encoding Gene Design and Protein Analysis

Rapid growth of global population should be demanded more high-quality food and this might be alleviated by improving protein nutrition levels in usual foodstuff. The expense associated with food additives used for animal production have led to the study of intracellular production of high-quality protein (HQP) by transgenic crops and other organisms, as a means of obtaining efficient and less costly sources of essential amino acids. One important approach is to extract high essential amino acids from bacteria or others. We designed a HQP gene encoding about 100 amino acids which had more than 80 % essential amino acids. GOR/Gibrat and Swiss-Pdb Viewer analysis were used to predict the secondary structure of HQP-1 in pI value, hydrophilic and hydrophobic properties. Then the HQP gene was transferred into

E. coli

BL21 (DE3), and the protein was extracted and identified by SDS-PAGE.

Guo-qing Huang, Lei Wang, Dong-kai Wang, Qiong Wu, Yao Li, Jin-hai Zhao, Di-fei Cao
Chapter 10. Isolation and Characterization of a Highly Siderophore Producing Bacillus subtilis Strain

Bacteria, fungi, and graminaceous plants synthesize siderophore for scavenging iron from environment to inhibit the growth of pathogens by depriving of iron. At this study, 19 siderophore producing bacterial strains were isolated from heat treated samples by O-CAS agar screening and liquid CAS assay. Among them, strain MB8 produced a relatively high level of siderophore (70.38 % SU, ++++). Comparative sequence analysis of 16S rRNA gene identified MB8 as a

Bacillus subtilis

strain. With Arnow analysis, the siderophore of MB8 was confirmed to be one type of catecholate. Furthermore, fermentation parameters affecting the siderophore production by MB8, including carbon sources and amino acids, were investigated at individual levels. Antimicrobial activity assay showed that the siderophore exhibited significant antimicrobial activity against

Proteus vulgaris, Staphylococcus aureus, Micrococcus luteus, Escherichia coli, Vibrio

parahaemolyticus

, and

Mucor

. Our results indicated that

B. subtilis

MB8 had the potential to be used as a probiotic organism.

Huiming Zhu, Hongjiang Yang
Chapter 11. Isolation and Identification of an Inulinase-Producing Strain and the Optimization of Its Fermentation Condition

Inulinases are classified among hydrolases, target the β-(2,1)-linkage of inulin and can be used to produce high-fructose corn syrup, fructo-oligosaccharides and alcohol, conferring upon inulinases significant potential for application in the food industry. A total of 28 strains that are capable of producing inulinase were isolated from the rhizosphere soil of

Jerusalem artichoke

(

Helianthus tuberosus

L.). Of these strains, the bacterial strain ni-3 was isolated through further screening. Based on its morphological, physiological and biochemical characteristics, ni-3 was identified by 16S rDNA sequences and systematic analysis. The 16S rDNA sequences of this strain had 99.5 % similarity with the partial sequence of

Brevibacillus centrosporus

, denominated as

B. centrosporus

ZF-9. The optimum conditions of fermentation were investigated. The optimum conditions for inulinase were as follows: inulin 45 g/L, yeast extract 11 g/L, and Na

2

HPO

4

20 g/L. Under these conditions, the inulinase activity of

B. centrosporus

ZF-9 reached 6.41 U/mL.

Yang Zhang, Hongyang Zhu, Jinhai Wang, Xiuling Zhou, Wei Xu, Haiying Shi
Chapter 12. Isolation and Identification of a Cellulose-Producing Bacterial Strain from the Genus Bacillus

Strain ZF-7 was isolated from defective fruits in this study, and its product was determined as bacterial cellulose in view of the results of cellulose special reaction, FTIR spectroscopy analysis, and cellulose hydrolysis experiments. Based on morphological, physiological, and biochemical characteristics, ZF-7 was identified by 16S rDNA sequences and systematic analysis. The results indicated that 16S rDNA sequence of ZF-7 had 99.5 % similarity to the partial sequence of

Bacillus amyloliquefaciens

, suggesting that ZF-7 was categorized as a species of

B. amyloliquefaciens

, and denominated as

B. amyloliquefaciens

ZF-7. The fermentation performance of ZF-7 under shaking and static flasks was inspected preliminary, and the BC production was 6.6 and 6.2 g/L, respectively.

Hongyang Zhu, Yang Zhang, Jinhai Wang, Yongning Li, Weiling Lin
Chapter 13. Improved Lactose Utilization by Overexpression β-Galactosidase and Lactose Permease in Klebsiella pneumoniae

As an important bio-based chemical product, 2,3-butanediol has a wide range of applications in many fields, such as chemical, fuel, food, and aerospace. Cheese whey powder (CWP), an inexpensive, available, and abundant material, is considered to be an ideal substrate for 2,3-BD fermentation. To improve 2,3-butanediol production, the previous studies mainly focus on the metabolic pathway from pyruvate to 2,3-butanediol or the metabolic pathway of by-products, but studies about improving lactose utilization rate are rarely reported. In the present study, adding exogenous β-galactosidase was proved to favor the lactose utilization and lactose utilization might be the limiting step of lactose fermentation to 2,3-butanediol.

ElacY

(encoding lactose permease of

Escherichia coli

) and

bgaB

(encoding β-galactosidase of

K. pneumonia

) were overexpressed in

K. pneumonia

CICC10781. Of the two genes, only overexpression of

ElacY

promoted lactose utilization of CICC10781, and meanwhile the 2,3-butanediol generation capacity was not affected.

Xuewu Guo, Yazhou Wang, Xiangyu Guan, Yefu Chen, Cuiying Zhang, Dongguang Xiao
Chapter 14. Breeding High Producers of Enduracidin from Streptomyces fungicidicus by Combination of Various Mutation Treatments

Enduracidin is a kind of lipodepsipeptides and composed by a 17 amino acid residual conjugated with a fatty acid strain. It is used as a feed additive approved by EU because of its high safety, low toxicity, low-residue, and activity against a wide variety of Gram-positive bacteria. Here, we combined a physical mutagenesis of

137

Cs γ-irradiation with the atmospheric and room temperature plasma (ARTP) biological breeding system to improve enduracidin productivity in the industrial Streptomyces. The mutation rates of spores treated by ARTP for 1 min and by

137

Cs γ-irradiation for 500 Gy dosages were 26.51 and 19.41 %, respectively. An optimal process for breeding by the combination was proposed. Through the high throughout screening method, the mutant

Streptomyces fungicidicus

S-224 was obtained, and its enduracidin yield reached to 1.58 g/L that was 1.65-fold to the original strain. The mutation breeding combined ARTP with

137

Cs γ rays was the first trial and provided an alternative method for industrial strains improvement.

Dong Zhang, Qingling Wang, Xinle Liang
Chapter 15. Expression of Stichopus japonicus Lysozyme Gene in Bacillus subtilis WB600

In this study, a genetic engineering bacteria

Bacillus subtilis

pHT43-SjLys/WB600 was successfully constructed for the expression of the lysozyme gene from sea cucumber (

Stichopus japonicus

) by the method of recombinant DNA technique. The growth trend of engineering bacteria was consistent with the wild-type strain WB600, and the results demonstrated that insertion of foreign gene did not affect its physiological and biochemical metabolism. In the absence of selection pressure, the analysis of the stability revealed that there was no gene rearrangement and lost of the recombinant plasmid in the bacteria which showed that it has high genetic stability. The SDS-PAGE results demonstrated that pHT43-SjLys/WB600 successfully expressed soluble SjLys protein after incubated for 48 h induced by ITPG. The heterologous expression protein of pHT43-SjLys/WB600 displayed remarkable inhibitive effect on the growth of the

Vibrio parahaemolyticus

. To our knowledge, this is the first report about the

SjLys

gene authentic heterologous expression in

B. subtilis

. It should provide a robust secretion expression system for genetic engineering of

B. subtilis

and was thus proposed a potentially new way for producing recombinant SjLys protein.

Zhiwen Liu, Xingyu Liao, Lu Sun, Dan Zou, Dan Li, Lina Cong
Chapter 16. Mega-Genome DNA Extraction from Pit Mud

Molecular analyses for the study of microbial community often depend on the direct extraction of the mega-genome DNA from pit mud. The present work compared six different methods to extract the mega-genome DNA from pit mud, and investigated the relationship between the quantity and the diversity of the mega-genome DNA. Using the six different extraction methods, the DNA ranged from 3.18 to 20.17 ng DNA/μL. Both the quantity and the diversity of the mega-genome DNA, the “repeated freezing and thawing” method is superior to other five methods, so it is the best choice for the extraction of mega-genome DNA from pit mud.

Huimin Xie, Yali Dai, Lin Yuan
Chapter 17. Evidence for a Link of SDPR and Cytoskeleton

Serum deprivation response (SDPR) is a plasma membrane binding protein and a substrate of protein kinase C (PKC) phosphorylation, which recruits polymerase I and transcript release factor (PTRF) to caveolae to stabilize and define their morphology. But how they were transported to membrane remain unclear. In order to clarify the link of SDPR and cytoskeleton, we observed the localized relationship between SDPR and cytoskeleton proteins by Immunofluorescence. Here, we discovered that SDPR colocalizes with cortactin (CTTN) in A7r5. Interestingly, SDPR was found to be highly concentrated in podosomes and dorsal ruffles induced by Phorbol 12, 13-dibutyrate (PDBu), and platelet-derived growth factor (PDGF), respectively, which suggest a possible role of SDPR in cell motility and cell invasion.

Baoxia Zhang, Jun Zhu, Liqiao Ma, Yuyin Li, Aipo Diao, Yinchuan Li
Chapter 18. CREB Regulated Transcription Coactivator 1 (CRTC1) Interacts with Microtubules

CRTCs are found to be coactivators of Creb1 that constitutively regulate cell energy balance and longevity by regulating glucose metabolism. To accomplish this, dephosphorylated CRTCs translocate to nuclear to enhance the activation of Creb1 to activate gluconeogenesis. Recently, CRTC1 was reported to be located in hippocampal neuron synapses and dendrites with long distance transport along neurites. In this report, we found that CRTC1 was distributed mostly in the cytosol along microtubules and was also able to target membrane ruffles, which suggests CRTC1 may relay signals far from cell periphery region and possibly directly from plasma membrane to cell nuclei. However, its mechanism is still unknown. Our result shows that CRTC1 binds to polymerized tubulins, which implies that signal transduction of CRTC1 complex may be mediated via microtubules. This finding helps to further understand the detailed role of CRTC1 in cell cytosol.

Liqiao Ma, Yu Sun, Baoxia Zhang, Yuyin Li, Aipo Diao, Yinchuan Li
Chapter 19. The Biological Effects of Carbon Nanotubes in Plasma Membranes Damage, DNA Damage, and Mitochondrial Dysfunction

In the last ten years, accompanied by deep development of nanotechnology, a large number of nanomaterials have come into people’s horizons. Carbon nanotubes (CNTs), one of the important nanometer materials, have been widely applied to various biomedical applications such as cancer photothermal therapy, specific drugs delivery, and so on due to their unique physicochemical property. Along with applications of CNT, the relationship between the biotoxicity of CNT and health was widely concerned. This review provides a general overview of the biological damage of overexposure of single-walled carbon nanotubes (SWCNTs) and mutiwalled carbon nanotubes (MWCNTs) and discusses some of the challenges associated with CNTs toxicity.

Zhuo Zhao, Zhi-Peng Liu, Hua Wang, Feng-Juan Liu, Hui Zhang, Cong-Hui Zhang, Chen-Guang Wang, Xiao-Chuan Jia
Chapter 20. Evidence of the Interplay of Menin, CRTC1 and THOC5 Triangles

Multiple endocrine neoplasia type 1 (MEN1) is an endocrine cancer syndrome occurred on multiple endocrine tissues caused by

MEN1

gene. Its protein product is 610 amino acids in length, named Menin. Creb-regulated transcriptional coactivator 1 (

CRTC1

) activates transcription through enhancing the interaction of CREB1 with TAF4. THOC5 was the fifth member of a subcomplex of the transcription/export complex (THO), which is involved in the nuclear export of a subset of mRNAs. In this report, we found an interesting regulation pattern between these three genes. CRTC1 promotes THOC5 nuclear export and Menin reverses this process. Both Menin and THOC5 inhibit CRTC1 activation on G6Pase promoter in a synergistic manner, suggesting the interplay of these three genes regulates gene expression.

Lichang Wu, Qiwen Zhang, Liqiao Ma, Yu Sun, Baoxia Zhang, Caicai Kang, Aipo Diao, Yinchuan Li

Optimization and Control of Biological Process

Frontmatter
Chapter 21. Classification of Lymphoma Cell Image Based on Improved SVM

Due to the diversity of lymphoma, its classification must rely on experienced pathologist in clinic pathologic analysis. In order to improve the accuracy of lymphoma classification, lots of image processing technologies and recognition methods were presented. The support vector machine (SVM) has been widely applied in medical image classification as an effective classification method. However, the application of SVM is blocked by the limitation that each classifier must adopt the same feature vector. In this paper, an improved SVM is proposed to overcome this limitation. Through the analysis of features of different classes, different feature vectors are obtained for each class of objects respectively. And then the improved SVM based on “one-against-one” strategy is applied to classify each class one by one. According to the results of classifying seven different lymphoma images, our classification method is effective to acquire the higher precision than conventional SVM and PSO-SVM model in lymphoma classification.

Ting Yan, Quan Liu, Qin Wei, Fen Chen, Ting Deng
Chapter 22. Foam Control in Epothilones Fermentation of Sorangium cellulosum

Eight different antifoam agents were added in shake flasks or in a 50 L fermenter, and the foam control abilities and effects on cultures were evaluated in this paper. Results showed that THIX-298 and SXP107 antifoam agents could eliminate foam rapidly and suppress foam during the fermentation process in the shake flasks. The biomasses and yields of epothilones were affected by different antifoam agents’ addition when 0.06 % (v/v) THIX-298 antifoam agent was added in the shake flasks. The yields of epothilone A and B had a significant increase of about 48.12 and 48.55 % compared to the control, respectively, representing the largest effects of the antifoam agents tested. Reducing sugar and amino nitrogen were also utilized more rapidly than the control. Additionally, efficiency parameters of the different antifoam agents were compared in the fermentor. Finally, THIX-298 antifoam agent was chosen as the optimal one.

Yue Liu, Lin Zhao, Hongrui Zhang, Fuming He, Xinli Liu
Chapter 23. Acute Toxicity by Four Kinds of Oil Dispersants in Cynoglossus semilaevis

This study evaluated the in vivo toxicity of four kinds of oil dispersants to

Cynoglossus semilaevis

, in acute exposures of 24, 48, 72, and 96 h through mortality. The 24 h LC

50

of RS-1, HH, GM-1, and FK were 17.0793, 4.6957, 3.2758, and 4.7510 mg/L, respectively. The 48 h LC

50

were 16.0173, 3.7251, 2.2135, and 2.5568 mg/L. The 72 h LC

50

were 14.6422, 2.9266, 1.9983, and 1.8877 mg/L. The 96 h LC

50

were 13.7879, 2.3060, 1.4391, and 1.5962 mg/L. The decreasing order of toxicity was GM-1 > HH > FK > RS-1. Compared with the grading table of toxicity of chemical dispersants, four kinds of oil dispersants were highly toxic.

Jinwei Gao, Wenli Zhou, Ruinan Chen
Chapter 24. Imprinted Cross-Linked Enzyme Aggregate (iCLEA) of Phenylalanine Ammonia Lyase: A New Stable Biocatalyst

The industrial use of phenylalanine ammonia lyase (PAL), an interesting biocatalyst for manufacture of L-phenylalanine (L-Phe) by reversing the enzyme reaction with high concentration of trans-cinnamic acids (t-CA) and ammonia, has been hampered by a lack of long-term stability and low activity toward substrates. In this study, it is shown that the PAL activity of such a CLEA can be improved by molecular imprinting with a suitable substrate. PAL was imprinted with t-CA and subsequently cross-linked with glutaraldehyde (iCLEAs). Compared to free PAL, PAL stability in the iCLEAs against substrate inhibition was significantly improved, furthermore, the iCLEAs exhibited good reusability. These results indicated that the procedure might be used as a feasible and efficient solution for improving properties of immobilized enzyme in industrial application.

Jian Dong Cui, Rong Lin Liu, Lian Lian Li
Chapter 25. Effects of Calcium on the Morphology of Rhizopus oryzae and L-lactic Acid Production

The effects of exogenous calcium on fungal pellet morphology during preculture and L-lactic acid production were studied. The results showed that addition of exogenous calcium could induce pellet formation. The diameter of the pellet increased with increasing concentration of exogenous calcium, including CaCl

2

and CaCO

3

. The smaller pellet precultured with low concentration of soluble calcium (CaCl

2

) was beneficial for L-lactic acid production because the pellet was dense and the large inner part of the pellet was inactive. By contrast, the larger pellet precultured with high concentration of insoluble calcium (CaCO

3

), except 8.0 g/L CaCO

3

, was beneficial for L-lactic acid production. Supported by the CaCO

3

powder, the entire biomass layer was fully active, and the highest L-lactic acid productivities of 1.22 g/L h and 58.6 g/L L-lactic acid were reached using the 1.5 mm pellet.

Yong-Qian Fu, Long-Fei Yin, Ru Jiang, Hua-Yue Zhu, Qing-Cheng Ruan
Chapter 26. Estimation of Dietary Copper (Cu) Requirement of Cynoglossus semilaevis Günther

To evaluate the dietary copper (Cu) requirement of

Cynoglossus

semilaevis

, inorganic Cu (0, 2, 6, 17, and 50 mg kg

−1

) was added to the basal diet, providing actual dietary Cu (5.91, 8.23, 11.78, 24.16, and 56.34 mg kg

−1

). Each diet was fed to

C. semilaevis

Günther (initial body weight, 68 g) in triplicate groups for 8 weeks in a flow-through system. The results showed that weight gain rate (WGR), specific growth rate (SGR), feed conversion rate (FCR), and protein efficiency ratio (PER) in fish supplemented with 11.78 mg kg

−1

were significantly higher than those in fish fed with the basal diet (

P

< 0.05), and no significant difference was observed in fish fed dietary Cu ranging from 11.78 to 56.34 mg kg

−1

(

P

> 0.05). The activities of protease, amylase, lipase, copper–zinc superoxide dismutase (Cu–Zn SOD), and lysozyme first increased and then decreased with the increasing dietary Cu concentrations, and 11.78 mg kg

−1

diet provided maximum activities. The optimum requirement of

C. semilaevis

for dietary Cu was estimated to be about 11–12 mg kg

−1

diet using broken-line regression analysis, based on the growth and enzyme activities.

Qingkui Wang, Yang Zhang, Dongqing Bai, Chengxun Chen, Yongjun Guo, Kezhi Xing
Chapter 27. Influence of Different Substrates on the Production of Pigments and Citrinin by Monascus FJ46

The aim of this work is to investigate the influence of various carbon sources, including cereals, tuber crops, and argo-industrial residues as substrates on the production of pigments and citrinin by Monascus FJ46. Compared with control, all the substrates can reduce the yield of citrinin except for glutinous rice flour and potato as substrates. The relative yield (citrinin concentration/red pigment valeur) with naked oats flour was 0.0012 μg/U, followed 0.0015 μg/U with millet flour, 0.0043 μg/U with sorghum flour, 0.0080 μg/U with corn flour, 0.0088 μg/U with cordyceps sinensis residues, and 0.0091 μg/U with sweet potato. Therefore, naked oats flour, millet flour, sorghum flour, corn flour, cordyceps sinensis residues, and sweet potato can be promising substrates for the production of pigments. Additionally, this is the first report on pigments production using cordyceps sinensis residues as substrate.

Hongxia Mu, Liubin Huang, Xuemei Ding, Shuxin Zhao
Chapter 28. FAD2B from a Peanut Mutant with High Oleic Acid Content Was Not Completely Dysfunctional

A peanut mutant with 72.53 % oleate content was selected from sodium azide mutagenized peanut Huayu 22, an export type cultivar with 51.65 % oleate content. The chemical mutant had a point mutation (C281T) in the coding region of

FAD2B

, which caused a T94I substitution in the FAD2B protein. Though the mutated

FAD2B

gene was incompletely dysfunctional as shown in yeast expression system, the peanut mutant has a high oleic acid phenotype. This indicated the possible involvement of other unidentified genetic factor(s) conditioning oleic acid content in peanut.

Xiu Zhen Wang, Qi Wu, Yue Yi Tang, Quan Xi Sun, Chuan Tang Wang
Chapter 29. Optimization of Sterilization Process After Spore Activation for Cereal Beverage in Large-Scale Production

Cereal beverage is a novel cereal drink made of whole grain. Grain seeds carry quantities of microorganisms, including spores difficult to be killed. In order to maintain its nutrition, stability, and palatability, sterilization technology of cereal beverage in a large-scale production was systematically investigated to obtain optimal sterilization process. Sterilizing strategy to kill all bacteria followed by spore activation was used for the first time to cereal beverage. Effects of activation temperature, activation time on total numbers of bacteria, spore and enduring-heating spore, pH value, and flavor freshness of cereal beverage were investigated. Optimal sterilization process after spore activation of large-scale production of cereal beverage was finally determined. Results showed that suitable temperature for spore activation was 65–85 °C, the total number of bacteria and spores in cereal beverage both decreased. Spore activation at 75 °C for 30 min, did not change the taste of fresh cereal beverage. After spore activation at 75 °C for 30 min, all the bacteria in cereal beverage could be killed at 134 °C for 16 s, however, microorganisms could be sterilized at above 140 °C for 16 s without preactivation. Finally, optimal sterilization parameters of cereal beverage in a large-scale production was set as UHT sterilization at 134 °C for 16 s after preactivation at 75 °C for 30 min.

Zhe Li, Liping Zhu, Shigan Yan, Junjie Liu, Wenjuan Zhao
Chapter 30. Optimization of Medium for Exopolysaccharide Production by Agaricus brunnescens

The exopolysaccharide of

Agaricus brunnescens

is a newly produced biomaterial for various applications. In this paper, we optimized the culture medium for this exopolysaccharide production by

A. brunnescens

using orthogonal methods. The optimal medium for enhancing production was determined as wheat bran 120 g/L, Glucose 25 g/L and peptone 2.0 g/L for exopolysaccharide yield. This optimization strategy in shake flask culture leads to an exopolysaccharide production of 12.13 g/L, which was considerably higher than that obtained in preliminary studies.

Li-tong Ban, Yu Wang, Liang Huang, Hongpeng Yang
Chapter 31. Effect of Attapulgite on Cell Activity of Steroid-Transforming Arthrobacter simplex

Attapulgite (AT) is a kind of nanostructure material. In this present study, the effect of AT on cell growth, permeability, and dehydrogenase activity of

Arthrobacter simplex

TCCC11037 (

ASP

) was investigated. The cell growth was significantly inhibited by adding AT to

ASP

medium. However, the cell growth increased when less than 0.1 % (w/w) of AT was added in the late logarithmic phase. The cell permeability increased with the increase of AT concentration, and the dehydrogenase activity of

ASP

significantly improved when the cell was treated by 0.3 % (w/w) of AT. Scanning electron microscope (SEM) analysis revealed that AT showed little effect on

ASP

cell integrity. This study provides the basic data for the practical application of AT in steroids biotransformation.

Yanbing Shen, Hengsheng Zhao, Yanhua Liu, Rui Tang, Min Wang
Chapter 32. Establishment of a Method to Measure the Interaction Between Nedd4 and UbCH5c for Drug Screening

Protein ubiquitination is closely associated with tumorigenesis. E3 ubiquitin ligase Nedd4 is overexpressed in prostate, bladder, and colorectal cancer. Nedd4 interacts with UbCH5c ubiquitin transferase through its HECT domain. Two GST-tagged Nedd4 proteins and His-HA-UbCH5c protein are expressed and purified. Using these recombinant proteins, a method is established to measure the interaction between Nedd4 and UbCH5c. This established method can be used to screen the small molecule inhibitors to block the interaction between Nedd4 and UbCH5c, which inhibit the activity of E3 ubiquitin ligase Nedd4.

Kunyuan Kou, Jianli Dang, Baoxia Zhang, Guanrong Wu, Yuyin Li, Aipo Diao
Chapter 33. Determination of Phthalate Esters in Tea by Gas Chromatography–Mass Spectrometry

A method was developed for the determination of five phthalate esters (PAEs) in tea. The phthalate esters were extracted from tea samples with an optimized extraction method and quantification was achieved by gas chromatography–mass spectrometry (GC–MS). The extraction parameters (i.e., extraction type, extraction solvent, extraction time, and amount of tea) and the conditions of detection were investigated and selected. Under the optimized conditions, the linearity of five PAEs were good in their detection range, the correlation coefficient of them were above 0.99. The limits of detection (LODs) were 0.24–3.72 μg/L. The spiked recovery varies from 87.63 to 98.08 %, and the relative standard deviations (RSD) were less than 8 % (

n

= 3). The proposed method was successfully applied to determine the five phthalate esters in commercial available tea samples, which suggested that the method was simple, less interference, and good repeatability.

Yan Lu, Liping Du, Yang Qiao, Tianlu Wang, Dongguang Xiao
Chapter 34. Antibacterial Mechanism of 10-HDA Against Bacillus subtilis

10-HDA (10-hydroxy-2-decenoic acid) is an unsaturated medium-chain fatty acid, which is the main active component of royal jelly. It has been shown to possess biological activity in antibacterial, immunoregulation, and antitumour. However, the underlying antibacterial mechanism of 10-HDA is unclear. In this study, it is shown that 10-HDA is a broad-spectrum antimicrobial agent and has obvious inhibition effects on multiple pathogenic bacteria with a concentration-dependent mode of inhibitory effect. The minimum inhibitory concentration (MIC) of

Bacillus subtilis

is 0.62 mg/mL. Furthermore, the acting mechanism of 10-HDA on

B. subtilis

is investigated by analyzing the DNA binding ability of 10-HDA with gel retardation assay and atomic force microscope. The results indicate that 10-HDA can combine with the bacterial DNA strongly and inhibit migration of DNA on Sepharose gel. By detecting the effect of 10-HDA on DNA content of bacteria, it was shown that 10-HDA had inhibitory effect on DNA synthesis of bacteria. Our results suggest that 10-HDA can combine with bacterial DNA, which further inhibits the DNA synthesis, and thus suppress the growth and activity of bacteria.

Xiaohui Yang, Junlin Li, Ruiming Wang
Chapter 35. Monitoring Glutamate and Glucose Concentration During the Temperature Triggered Glutamate Fermentation by Near-Infrared Spectroscopy

In this study, the calibration models for monitoring concentrations of glutamate and glucose in the temperature-triggered glutamate fermentation process were developed by near infrared (NIR) spectroscopy. The NIR measurements of samples were analyzed by partial least-squares (PLS) regression with selecting spectral pre-processing methods and different wavelengths. The root-mean square errors of cross-validation (RMSECV) of glutamate and glucose were 2.73 and 1.92 g/L, respectively. The determination coefficients (R

2

) were 0.996 and 0.982, respectively. The residual predictive deviation (RPD) was 17.8 and 8.37, respectively. These results showed that all models had good predictive ability. New batch fermentation as an external validation was used to check the models. Compared with concentrations of predict value and measured value, the determination coefficient was 0.992 and 0.951, respectively. The average relative errors were 5.79 and 7.38 %, respectively. These results showed that prediction model could predict and monitor the temperature-triggered glutamate fermentation process accurately and quickly, and thus theoretical basis for the real-time control and optimization in the temperature-triggered glutamate fermentation process was provided.

Yongli Gui, Jingbo Liang, Chenglin Zhang, Xixian Xie, Qingyang Xu, Ning Chen, Lei Ma
Chapter 36. Effect of Sodium Citrate on L-tryptophan Fermentation by Escherichia coli

L-tryptophan is an essential amino acid, and the work for L-tryptophan production by microbial fermentation has important practical significance with the development of L-tryptophan market. In this study, according to the principle of metabolic engineering, the medium of L-tryptophan fermentation by the strain

Escherichia coli

TRTH was optimized. The accumulation of by-products was decreased and production of desired product was increased by adding sodium citrate to medium. The effect of sodium citrate on L-tryptophan fermentation was investigated in a 30 L fermentor. The results indicated that when L-tryptophan fermentation with the medium containing 2 g/L sodium citrate, as compared with the medium no containing sodium citrate, the biomass, L-tryptophan production and glucose conversion rate were increased by 2.57, 5.32, 4.21 %, and the accumulation of acetate, pyruvate and lactate were decreased by 5.15, 4.89, 5.23 %. With the medium containing 2 g/L sodium citrate for L-tryptophan fermentation, the biomass, L-tryptophan production and glucose conversion rate were 42.7, 35.7 and 18.2 % respectively.

Qing-yang Xu, Li-kun Cheng, Xi-xian Xie, Cheng-lin Zhang, Yan-jun Li, Chen Ning
Chapter 37. Reduction Reaction of Methyl Condensation Compound by Saccharomyces cerevisiae

Microbial transformation of 13β-alkyl-3-methoxy-8, 14, secogona-1, 3, 5(10), 9-tetraen-14, 17-dion with

Saccharomyces cerevisiae

resulted in two metabolites: product 1 (P1) and product 2 (P2). Among these, P1 was identified to be 13β alkyl-3-methoxy-8, 14-seco-1, 3, 5(10), 9(11)-estratetraene-17β-ol, 14-one, which has been studied extensively. P2 was purified, crystallized, and identified as 13β-alkyl-3-methoxy-8, 14, secogona-1, 3, 5(10), 9-tetraen-14, 17-diol by X-ray single crystal diffraction method. It was first reported in fermentation medium and the dihydroxyl reduction is a new reaction in biotransformation of steroid.

Lu Yu, Shuhong Mao, Shaoxian Ji, Xiaoguang Liu, Fuping Lu
Chapter 38. Study on Ultrasonic-Assisted Extraction of Essential Oil from Cinnamon Bark and Preliminary Investigation of Its Antibacterial Activity

Ultrasonic-assisted extraction of essential oil from cinnamon bark was explored in this study. Effects of different parameters such as solvent, extraction time, powder granularity, ultrasonic power, solid–liquid ratio, and extraction temperature on the yield of essential oil were also investigated through single factor and orthogonal tests. The antibacterial activity of cinnamon oil obtained by ultrasonic-assisted extraction was preliminary determined. Results show that ultrasonic power has the largest effect on the oily yield, followed by powder granularity, time, and solid–liquid ratio. The optimized extraction conditions are as follows: petroleum ether (b.p. 60–90 °C) as extraction solvent, 165 W, 80–100 mesh cinnamon power, 40 min, and 6:60 (W/V) solid–liquid ratio, respectively. And under this condition, cinnamon oil yield can reach 14.8034 %, in which the content of trans-cinnamaldehyde is 82.62 % by HPLC analysis. Ultrasonic-assisted extraction of essential oil from cinnamon bark is feasible, and it can be used as a good alternative compared with other traditional methods. In addition, antibacterial activity assay demonstrates that cinnamon oil shows a strong inhibitory effect against

Escherichia coli

and

Bacillus subtilis

, and it can be applied as a natural antibacterial agent in many areas.

Ping Li, Lin Tian, Tao Li
Chapter 39. Geranyl Butyrate Production by Candida antarctica Lipase B-Displaying Pichia pastoris

Enzyme-displaying whole-cell yeast is becoming an alternative to immobilized enzyme. Here, a recombinant

Pichia pastoris

displaying Candida antarctica lipase B (CALB) was used to catalyze the synthesis of geranyl butyrate with geraniol and butanoic acid as substrates in n-heptane. Response surface methodology (RSM) with a four-variable five-level central composite design (CCD) was applied when biocatalyst concentration, geraniol concentration, butanoic acid/geraniol molar ratio, and water activity were taken as influential factors to improve the production of geranyl butyrate. The results indicated that both substrates molar ratio and water activity affected significantly the geranyl butyrate yield (

p

< 0.05). Biocatalyst concentration 20.9 g/L, geraniol concentration 0.57 mol/L, butanoic acid/geraniol molar ratio 1.21:1, and water activity 0.53 were the optimum of the ester synthesis. Under the above conditions, the geraniol conversion rate was 98.5 % in 10 mL reaction after 9 h reaction, which was equivalent to 126 g/L geranyl butyrate. When the esterification reaction was scaled up in batch stirred reactors, 97.87 % of geraniol conversion rate in 200 mL as well as 97.56 % in 500 mL could be obtained.

Zi Jin, Janvier Ntwali, Ying Lin, Huang Kui, Suiping Zheng, Shuangyan Han
Chapter 40. Metabolic Analysis of a Corynebacterium glutamicum IdhA Mutant During an Efficient Succinate Production Using pH-Control Under Oxygen Deprivation

Corynebacterium glutamicum

produce succinate and lactate from glucose under oxygen deprivation without growth. By deleting the lactate dehydrogenase genes (

ldhA

), lactate formation could be completely abolished and yield of succinic acid increased up to 0.78 g g

−1

. With increasing concentration of carbonate, which could regular the pH of culture, the glucose consumption rate has been improved, leading to increase the succinic acid production rates. Interestingly, depending on the pH conditions, there is a sharp increase in the presence of higher concentration carbonate. This phenomenon was investigated by the key metabolic enzymes analysis during different pH condition. A pyruvate carboxylase and malate dehydrogenase played a significant role in succinic production under oxygen deprivation. Succinic acid production by a

ldhA

mutant was investigated with free pH and constant pH 8.0. Succinic acid production and yield were higher under constant pH 8.0 conditions compared with culture with free pH. In an efficient succinic acid production process,

C. glutamicum

ATCC 13032 Δ

ldhA

accumulated 67.2 g l

−1

succinic acid within 48 h with an average succinic production rate 0.14 g l

−1

per cells (dry weight) (cdw) per hour.

Chen Wang, Heng Cai, Zhihui Zhou, Hong-gui Wan, Ping-kai Ouyang
Chapter 41. Effects of Stimulators on Lutein and Chlorophyll Biosyntheses in the Green Alga Chlorella pyrenoidosa Under Heterotrophic Conditions

In this study, the effects of some stimulators on lutein and chlorophyll biosyntheses in dark grown

Chlorella pyrenoidosa

were investigated. The aim of this study was to find out some stimulators that could effectively stimulate lutein and chlorophyll biosyntheses in

C. pyrenoidosa

in darkness. Vitamin A showed a profound effect in stimulating lutein biosynthesis, 70.9 % of increase of lutein content was observed when 4 mM vitamin A was supplemented in the culture. Similarly, chlorophyll biosynthesis was also improved by vitamin A. In addition, β-carotene and zeaxanthin also showed a positive effect on lutein accumulation in

C. pyrenoidosa

under heterotrophic conditions. The lutein content increased by 27.1 and 19.8 % when 0.1 mg/mL zeaxanthin and β-carotene were added into the culture, respectively. Besides, added zeaxanthin also significantly improved the lutein yield, by 45.4 %. In conclusion, lutein biosynthesis could be effectively stimulated by the selected stimulators in the green alga

C. pyrenoidosa

under heterotrophic conditions.

Tao Li, Dongqing Bai, Lin Tian, Ping Li, Yihan Liu, Yue Jiang
Chapter 42. Pharmacophore-Based Virtual Screening and Result Analysis of Histone Methyltransferase SMYD3 Inhibitors

Histone methyltransferase SMYD3 is a kind of protease relating to different kinds of tumors, which makes it a promising target for anticancer drugs. In order to find new small molecule inhibitors of SMYD3 for oncotherapy, a pharmacophore was created based on the crystal structure of SMYD3 with sinefungin (SFG) from Protein Data Bank (PDB ID: 3PDN) by using the software of Ligand Scout 3.1. The virtual screening of ZINC database was conducted with the pharmacophore using MOE software. Two hundred and four hits which meet at least five pharmacophore characteristics were obtained and four structural characteristics were summarized from these hits, which have laid a data foundation for the discovery of novel SMYD3 inhibitors.

Shaodan Liu, Ziyue Sun, Yonghui Zhong, Qingxin Cui, Xuegang Luo, Yujie Dai
Chapter 43. Effects of K2HPO4 on the Growth of Nostoc Flagelliforme in Liquid Media with Different Carbon Sources

Liquid fermentation is a promising strategy for sustainable production of

Nostoc flagelliforme

cells. In order to elucidate whether phosphate could promote the growth of

N. flagelliforme

in liquid fermentation, the effects of K

2

HPO

4

on

N. flagelliforme

growth were investigated in liquid media with 15 mM NaHCO

3

and 20 % CO

2

(v/v), respectively, in this study. Results showed that K

2

HPO

4

could promote

N. flagelliforme

growth under culture conditions with both kinds of carbon sources, respectively. The optimal concentration of K

2

HPO

4

for growth and protein contents of cells is 40–60 mg/L for the existence of each carbon source. The maximum dry cell weights of 0.75 g/L and 1.19 g/L were achieved in media with 15 mM NaHCO

3

and with 20 % CO

2

, respectively, when the concentration of K

2

HPO

4

is 60 mg/L. In media with 15 mM NaHCO

3

, the photosynthetic and respiration rates’ uptake of nutrients and protein contents increased significantly when K

2

HPO

4

concentration increased to 60 mg/L and then reached a plateau, however, higher concentrations of K

2

HPO

4

inhibited EPS accumulation. By contrast, in media with 20 % CO

2

, all growth characteristics above of

N. flagelliforme

were promoted, and reached a plateau at higher K

2

HPO

4

concentrations. These results provide valuable information for the cultivation of

N. flagelliforme

cells.

Hexin Lv, Feng Xia, Shiru Jia, Xianggan Cui, Nannan Yuan
Chapter 44. Production of Alkyl Polyglucoside Using Pichia pastoris GS115 Displaying Aspergillus aculeatus β-Glucosidase I

Pichia pastoris

GS115 displaying

Aspergillus aculeatus

β-glucosidase I was used as whole-cell biocatalyst catalyzing the synthesis of 1-hexyl glucoside, 1-octyl glucoside, and 1-decyl glucoside through reverse hydrolysis in water–alcohol two-phase system, while glucose and primary alcohols were used as substrate. In this study, factors affecting the synthetic yield were optimized in our study, including water content, glucose concentration, whole-cell biocatalyst content, the pH value of sodium acetate buffer, and the reaction temperature. In 5 mL reaction volume, the optimal reaction conditions of HG were: 10 % pH 3.0 buffer, 0.2 mol/L glucose, 10 g/L whole-cell biocatalyst; the optimal reaction conditions of DG: 15 % pH 3.0 buffer, 0.6 mol/L glucose, 10 g/L whole-cell biocatalyst; the optimal reaction conditions of DG: 20 % pH 2.0 buffer, 0.2 mol/L glucose, 40 g/L whole-cell biocatalyst; three reactions were conducted under 55 °C. After 72 h the maximum yield of HG was 11.5 %, that of OG and DG were 5.3 and 3.2 % after 96 h, respectively. The reutilization experiments inducted that the whole-cell biocatalyst could be used at least for five times without obvious decrease in synthesis ability.

Yajun Kang, Binru Wei, Dongheng Guo, Suiping Zheng
Chapter 45. Enhancement of Gellan Production in Sphingomonas paucimobilis JLJ by Heterogeneous Expression of Vitreoscilla Hemoglobin

A heterologous expression of

Vitreoscilla

hemoglobin (VHb) for improving cell growth and fermentation products has been successfully demonstrated in various hosts. To improve a commercially used strain for gellan, vgb gene was expressed in

Sphingomonas paucimobilis

JLJ under the control of the oxygen-dependent vgb gene promoter (P

vgb

). The promoter was maximally induced under microaerobic conditions. Biochemical activity of expressed vgb was confirmed by PCR, SDS–PAGE, and the CO-difference spectra analysis which exhibited a characteristic absorption maximum near 420 nm. The results showed that the expression of vgb could enhance cell growth. The composition of fermentation medium and the physical conditions of cultivations were investigated. The optimum medium consists of (g/L): Sucrose 30, Soybean meal 5, NaNO

3

1, KH

2

PO

4

1, K

2

HPO

4

1.5, MgSO

4

0.6. The optimum fermentation conditions were confirmed: pH 6.0, seed age 16 h, inoculum volume 10 %, 200 rpm, 30 °C, and the maximum gellan yield of vgb

+

recombinant was 1.713 % ± 0.02 % after cultivated 72 h, 21 % higher than that of the parental strain. VHb exhibited positive effect on cell growth and optimize gellan yield. The results provided potential applications of vgb gene in other industrially important strains to solve the increasing demand of aerobic processes in polysaccharides production.

Qinglong Ji, Dan Li, Xiangqian Li, Ting Li, Lin Yuan
Chapter 46. Enhanced Adenosine Production by Bacillus subtilis at Condition with Comprehensively Controlled Dissolved Oxygen and pH During Fermentation

Adenosine has potent effects on cardiovascular diseases and has been widely used as an antiarrhythmic agent. To improve its production, we investigated the effects of dissolved oxygen (DO) and pH on production by

Bacillus subtilis.

Based on the kinetic parameters at different DO levels, we proposed a two-stage DO strategy to control DO level at 30–40 % before 20 h of fermentation and 10–20 % after 20 h of fermentation, and confirmed that using this strategy could increase adenosine yield to 19.2 g/L in 52 h, which is increased by 78.6, 66.7, 9.5, 18.6, and 32.2 %, compared to the conditions with DO uncontrolled or controlled at 0–10, 10–20, 20–30, and 30–40 %, respectively. On this basis, pH was adjusted to further boost adenosine production. The results showed that the two-stage DO plus pH-shift method was the optimal way for adenosine production, under which, adenosine yield was further improved by 21.4 %, reaching 23.3 g/L at 56 h. To our knowledge, this is the first report on adenosine production verifying that the two-stage DO plus pH-shift method is effective for enhancing adenosine yield by

B. subtilis

.

Yue Liu, Juhua He, Qingyang Xu, Chenglin Zhang, Ning Chen, Xixian Xie
Chapter 47. The Semi-continuous Cultivation of Nostoc flagelliforme Cells

The semi-continuous cultivation is an important technique for large-scale liquid photoautotrophic cultivation of microalages, in which the excessive cells can be removed and the nutrient elements in the culture medium can be supplemented in time. In this paper, the semi-continuous cultivation of

Nostoc flagelliforme

cells was conducted and the cultivation conditions were optimised in order to achieve maximum biomass production. After single factor and Box-Behnken experiment with 15 days, the largest biomass amount (0.305 g/l) can be harvested at the best culture conditions (update rate 33 %, update cycle 1 day, and sodium nitrate concentration 1.6 g/l). This study offered important basic data for the implementation of large-scale photoautotrophic cultivation of

N. flagelliforme

cells.

Lifang Yue, Yupeng Xiao, Guojuan Sun, Shiru Jia, Yujie Dai, Xing Zheng
Chapter 48. Study on Ecological Diversity of Pectase and Its Producing Strains

Pectase is a heterogeneous group of related enzymes that hydrolyze the pectic substances. In this paper, seven strains producing high activity of alkaline pectate lyase were screened from alkaline soil and then the classification and activities of pectase from all these stains were detected. The results showed that seven strains had enzyme activity of pectinesterase and pectate lyase, but strains 410, 418, and 601 produced protopectinase, and its activity reached 0.341, 0.51, 0.28 u/mL separately.

Bacillus

sp. 410 and 418 showed pectin hydrolysis enzymes activity of 0.035 and 0.363 u/mL. When tested by pectin with 60 % esterification, only

Bacillus

sp. 520 had hydrolyzes activity. All these show that the pectase is abundant in different strains. Analysis of pectase system can provide fundamental basis for the study of application of wild strain, construction, and evaluation of engineering bacteria.

Jing Xiao, Xiwang Zhou, Xiaolong Zhang, Ruiming Wang

Biological Separation and Biological Purification

Frontmatter
Chapter 49. The Extraction and Regeneration of Resin XAD-16 in the Purification of Epothilones

The XAD-16 resins, which can capture the epothilones in situ and remove them from the culture broth, are always used in isolation and purification of epothilones during the fermentation process. In this paper, the effects of five different organic solvents (methanol, alcohol, acetone, ethyl acetate, carbon tetrachloride) on the epothilone extraction and regeneration of macroporous adsorption resin XAD-16 were surveyed. Of five organic solvents, methanol showed a much better effect on epothilone yield than the others. Also, the differences in structure between the fresh-resin and the regenerative-resin with methanol by atomic force microscope were analyzed. As the results, pore size of methanol-resin was larger than fresh-resin’s, which lead it more conducive to the adsorption of epothilones and regenerative-resin treated with methanol was sturdier. Result of the impact experiment showed the macroporous adsorption resin XAD-16 could be used for at least five times under methanol treatment.

Can Li, Lin Zhao, Xiaona Wang, Qiang Ren, Xinli Liu
Chapter 50. Conversion Process of High Color Value Gardenia Red Pigment

Pretreatment conditions of geniposide and conversion process conditions of gardenia red pigment were optimized, gardenia red pigment was refined by acid precipitation or resin, and the effects of refining were compared, conversion process of high color value gardenia red pigment was studied, the results suggested that 5 g sodium hydroxide was added to the 50 mL concentrated geniposide at 40 °C and stirred for 3 h, 12.5 g citric acid was added with stirring until it was dissolved, then 30 mL pretreated geniposide and 6 g sodium glutamate were added to 50 mL fermentation broth of

Aspergillus niger

, it was kept at 50 °C in vacuum incubator for 25 h, then the fermentation broth was put into the boiling water for 2 h. Under the above conditions, the color value of gardenia red pigment which refined by acid precipitation or resin was 53 and 63, respectively. It was improved significantly when compared with color value was 4 under the initial conditions.

Shangling Fang, Wei Jiang, Jinghua Cao, Xu Xu, Yanyan Jing, Maobin Chen
Chapter 51. Efficient Purification and Active Configuration Investigation of R-phycocyanin from Polysiphonia urceolata

An efficient method for purification of R-phycocyanin (R-PC) from

Polysiphonia urceolata

using anion exchange chromatography with pH elution combined with ammonium sulfate precipitation was proposed. R-PC was conveniently purified on a DEAE-Sepharose Fast Flow chromatography column with elution of pH 4.8 eluants after ammonium sulfate precipitation. The R-PC obtained was pure and with a recovery of 38.83 %. The absorption maxima of the purified RPC were at 550 and 618 nm, and the fluorescence emission maximum was at 632 nm with an exciting wavelength of 580 nm. The aggregation state of R-PC was (αβ)

3

and the molecular weights of the α and β subunits were 16.7 and 18.9 kDa, respectively. The efficient method has the potential to be applied in the R-PC commercial production. The active configuration investigation showed that RPC presented a good structural stability in the pH range of 4–9.

Li-ping Zhu, Shi-gan Yan, Ai-jie Lv
Chapter 52. Concentration of Sinigrin from Indian Mustard (Brassica juncea L.) Seeds Using Nanofiltration Membrane

Sinigrin has drawn more attention of pathologists and immunologists, which exhibits a wide range of biological activities. In the present study, the feasibility of concentration of sinigrin using nanofiltration membranes was studied using one-factor-at-a-time method with the permeate flux and sinigrin retention as responses. After seven commercial membranes were compared, including NF, NF-270, SPESI, STARMEN 120, STARMEN 128, STARMEN 220, STARMEN 240, we investigated the effects of temperature, pressure, and feed concentration on the concentration process of sinigrin from crude aqueous extracts of Indian mustard (

Brassica juncea

L.) seeds followed by primary ultrafiltration. The results indicated that NF-270 membrane showed the best concentration capability to sinigrin. Their ranges of permeate flux and sinigrin retention were 7.44–23.47 L/(m

2

h) and 90.77–97.13 %, respectively. Furthermore, the high flux and high retention were explained by swelling ratio, SEM, and ATR-FTIR, which were applied to determine the differences of nanofiltration membrane before and after soaking in aqueous solution. Finally, it could be concluded that the concentration process using nanofiltration provided a novel, rapid, and economical method for concentration of sinigrin.

Tianxin Wang, Hao Liang, Qipeng Yuan
Chapter 53. Optimization of Crude Polysaccharides Extraction from Dioscorea esculenta by Response Surface Methodology

In this study, response surface methodology (RSM) was employed to optimize the extraction process of crude polysaccharides from

Dioscorea esculenta

(lesser yam) performed using water decoction. Response surface methodology, based on a three-level, three-variable central Box–Behnken design (BBD), was employed to obtain the best possible combination of extraction time (

X

1

: 2.0–3.0 h), extraction pH (

X

2

: 9–11), and water to the raw material ratio (

X

3

: 20–30 mL/g) for maximum polysaccharide extraction. The experimental data obtained were fitted to a second-order polynomial equation using multiple regression analysis and also analyzed by appropriate statistical methods (ANOVA). The optimum extraction conditions were as follows: extraction time of 2.66 h, extraction pH of 10.07, and the ratio of water to raw material of 26.36 mL/g. Under these conditions, the experimental yield was 4.92 ± 0.037 %, which is well in close agreement with the value predicted by the model 4.96 %.

Kaihua Zhang, Liming Zhang, Na Liu, Jianheng Song, Shuang Zhang
Chapter 54. Nanofiltration Extraction and Purification Method for Cyclic Adenosine Monophosphate (cAMP) from Chinese Date Fruit

C

yclic adenosine monophosphate (cAMP) is a kind of important bioactive substances. The cAMP content of Chinese date fruit is particularly abundant and stable, more than any of the other plants researched so far. In this chapter, we report the development of nanofiltration extraction method for cAMP from date fruit (jujube) paste. The paste was hydrolyzed at 50 °C with 2.5 g/L pectinases (pectin lyase, cellulose, and hemicellulose) for 3 h. The yield efficiency of date juice achieved 70 %, and most importantly, the extracted cAMP content increased from 10.8 to 15.7 mg/100 g. The results prove that pectic substances have a protective effect to ring adenosine phosphate After resin adsorption, elution with formic acid, and filtration through a 150–500 Da Nanofiltration membrane, the crude cAMP content was increased to 36 %. The purification percentage of cAMP using silica gel column chromatography was as much as 97 %. The date fruits were soaked and pureed, and then subjected to enzyme hydrolysis, filtration, resin adsorption, formic acid elution, and nanofiltration. Pure cAMP was obtained after purification via a silica gel chromatography column. The byproduct of cAMP extraction could be further developed, which might enable a nonpolluting industrial chain of products.

Chunxia Wang, Yihan Liu, Hongbin Wang, Lianxiang Du, Fuping Lu
Chapter 55. Effect of Manchurian Walnut Extracts on Cancer Cells Proliferation

Extracts from the Manchurian walnut have the antibacterial, anti-inflammatory, and anticancer properties. In this study, different extracts were obtained from Manchurian walnut peels, flowers, and leaves by the Soxhlet extraction method, and used to study the antitumor activity. MTT assay shows that the ethyl acetate phase of the peels inhibits cell proliferation of colon cancer cell SW480 with an IC

50

at 56 μg/mL. Hoechst 33258 staining illustrates that the ethyl acetate phase of the peels induces cell apoptosis of SW480 cells. These results indicate that the ethyl acetate of Manchurian walnut peels inhibits cell proliferation and promotes cell apoptosis of cancer cells.

Changcai Zhao, Xing Niu, Rui Huang, Jiali Dong, Yuyin Li, Aipo Diao
Chapter 56. Extraction and Purification of Lumbrokinase from the “Ohira the 2nd” Earthworm

Lumbrokinase is an ideal drug hypertension to cardiovascular diseases. This essay discussed the purification process of lumbrokinase and decided its molecular weight with “Ohira the 2nd” earthworms as raw material. After ammonium sulfate precipitation, the crude enzyme was prepared, which was carried through chromatography of Sephadex G-75 and then DEAE-52. Certain high-purity lumbrokinase components were obtained, whose protein molecular weight was 33KD and specific activity was 1,784 U/mg, after SDS-PAGE test. These lumbrokinase components showed a direct fibrinolytic and plasminogen activation dual role, and a strong thermal stability and pH stability, which was good to thrombolysis.

Tianjun Li, Jian Ren, Tao Li, Yingchao Wang
Chapter 57. Study on Green Crystallization Process for L-glutamic Acid Production

To investigate the production of L-glutamic acid with high yield as well as improved purity, the optimization of crystallization process was conducted. During this study, various physicochemical parameters (e.g., initial temperature, cooling rate, acid adding rate, ultrasonic time, and stirring speed) of concentrated isoelectric crystallization method were evaluated to optimize the yield and purity of L-glutamic acid. The optimum crystallization parameters are as follows: acid adding rate 0.5 mL/min, ultrasonic time 10 min, and stirring speed 200 rpm. The yield of L-glutamic acid at optimal condition was 95.4 %, attaining a 6.5 % growth. The purity of crystallized product exceeded 99 %, giving a rise of 4 %. The optimal crystallization process with higher yield and improved purity reduces the energy consumption and thus promotes sustainable development.

Zhi-hua Li, Cheng-lin Zhang, Qing-yang Xu
Chapter 58. Synthesis of Poly (β-L-malic Acid) by the Optimization of Inorganic Nitrogen Complexing with Growth Factors Using Aureobasidium pullulans CGMCC3337

Poly (β-L-malic acid) (PMLA) can be used as a prodrug or used for drug delivery system, and it has attracted industrial interest. In previous studies, we could obtain 38 g/L PMLA with 10 g/L yeast extract as a nitrogen source, but the complex broth component made it difficult to the downstream process. To decrease the pressure in the extraction process, we used inorganic nitrogen resources and growth factors to replace yeast extract. Response surface methodology (RSM) was applied to optimize the fermentation medium formulation to improve the production of poly (β-L-malic acid) (PMLA) with

Aureobasidium pullulans CGMCC3337

. It was confirmed that ammonium nitrate 2.06 g/L, adenine 0.02 g/L, cytosine 0.012 g/L aspartate (Asp) 0.42 g/L, histidine(His) 0.6 g/L, leucine(Leu) 0.4 g/L, and threonine(Thr) 0.28 g/L could replace the yeast extract in PMLA production. The model developed based on RSM successfully predicted PMLA productivity (

R

2

= 0.9697). With the optimum medium established, 37.62 g/L PMLA was accumulated in the fermentation broth, and the impurities in the fermentation broth was reduced apparently.

Changsheng Qiao, Yumin Song, Zhida Zheng, Xujia Fan, Shiyun Yu
Chapter 59. Primary Characterization and In Vitro Antioxidant Activities of Polysaccharides from Yam Peel

The research purpose is to make full use of yam peel resources. In this present study, yam peel polysaccharide (YPP) was isolated from the

Dioscorea esculenta

peels. The primary characterization of YPP was explored, and we carried on the in vitro antioxidant experiment research. The FT-IR analysis was performed. And the absorptions at around 1,148 cm

−1

indicated a pyranose form of sugars. In vitro, YPP exhibited a potent scavenging activity on the hydroxyl radical, DPPH radical and superoxide radical, especially scavenging activity for DPPH radicals. The IC

50

value of YPP for eliminating hydroxyl radicals, DPPH radical, and superoxide anion radicals was respectively 9.5, 2.1, and 10.2 mg/ml. And the highest inhibition rates were, respectively, 60.2, 79.3, and 54.0 %. In this experiment, antioxidation test in vitro indicated that YPP was similar to other yam tuber polysaccharides. Furthermore, the further works on purification and isolation studies were done and the primary characterization of YPP were shown by chromatography. In general, these results suggested that the YPP may be explored as a novel and effective natural antioxidant to alleviate oxidative stress, and they may find promising applications as potent antioxidant agents and antiaging medicines.

Na Liu, Liming Zhang, Kaihua Zhang, Aiying Tian, Ruichao Li
Chapter 60. Optimization of Sample Preparation for the Metabolomics of Bacillus licheniformis by GC-MS

Metabolomic has become an important method in microbiology study. Sample preparation affects the quality of final metabolomics analysis strongly. There is no universal preparation method that can suit the metabolomics study of all kinds of microorganism, because of their various cell structure. In this study, a suitable sample preparation method for the metabolomics study of

Bacillus licheniformis

was explored and optimized. The main steps in metabolite sample preparation include quenching, estimation of leakage, and metabolite extraction. The result indicated that 60 % methanol, 0.9 % ammonium carbonate buffer was an appropriate quenching solution for

Bacillus licheniformis

by measuring intracellular metabolites, energy charge, and intracellular metabolites. Among the four different extraction methods (cold pure methanol, PM; cold methanol/water (70:30 v/v), MW; acetonitrile/methanol/water, (2:2:1 v/v/v), AMW; or acetonitrile/water (1:1 v/v), AW), MW was superior to others on the intracellular metabolites, which could effectively extract more intracellular metabolites. The results imply that the optimized preparation method for

Bacillus licheniformis

is critical for a reliable and accurate analysis of metabolome.

Hongbin Wang, Zhixin Chen, Jihan Yang, Yihan Liu, Fuping Lu
Chapter 61. Characterization of Recombinant L-Amino Acid Deaminase of Proteus mirabilis

L-amino acid deaminases catalyze the deamination of L-amino acids. Up until now, two types of L-amino acid deaminase have been identified in

Proteus

species. To investigate enzymatic characteristics of L-amino acid deaminase from

Proteus mirabilis

, L-amino acid deaminase encoding gene (

pmta

) was cloned from

P. mirabilis

T-1. Prokaryotic expression system was established to express recombinant Pmta. Enzymatic characteristics of the enzymes were analyzed. Results showed that recombinant Pmta exhibited function of second type of L-amino acid deaminase. The Km and Vmax value of Pmta for histidine was 10.57 mmol/L and 202.06 μmol/min/mg, respectively. The optimal temperature and pH of recombinant Pmta was 40 °C and 7.0. The enzymatic characteristics of Pmta were different from those of Pm1 discovered in

P. mirabilis

KCTC, which was probably due to different amino acid sequences. The Pmta deaminase will be very useful in the preparation of commercially valuable materials including urocanic acid and 3-mercaptopyruvic acid.

Chenglin Zhang, Jia Feng, Xixian Xie, Qingyang Xu, Ning Chen
Chapter 62. Screening for Strains Capable of 13β-ethyl-4-gonene-3, 17-dione Biotransformation and Identification of Product

Screening of strains for improvement in the yields of the desired steroid as well as production of novel steroid is highly demanded. In this study, a filamentous fungus

Aspergillus oryzae

capable of transforming steroid efficiently was screened from the collection of fresh bark of south China. A novel transformation product was purified, crystallized, and determined as [13, 17β-]furan-17-hydroxyl-4-gonene-3one by single-crystal X-ray diffraction. At the end of the transformation, the yields of the product reached to 95 %, which indicated

A. oryzae

can be efficiently applied in the field of steroid biotransformation with the novel biocatalytic ability.

Linlin Huang, Xiaoguang Liu, Yulan He, Pingping Wei, Shuhong Mao, Fuping Lu
Chapter 63. Extent and Pattern of DNA Cytosine Methylation Changes Between Non-pollinated and Pollinated Ovaries from Cymbidium hybridium

In this study, MSAP (methylation sensitive amplified polymorphism) technique was carried out to analyze differences in the methylation status between ovaries before and after pollination from

Cymbidium hybridium

. 72 selective primer combinations were used to check the status of DNA cytosine methylation and a total of 5,892 fragments were obtained. The results demonstrated DNA methylation events occurred in ovaries from

C. hybridium.

Both total and full methylation levels (14, 9.5 %) in the pollinated ovaries were lower than those in non-pollinated ovaries (11.4, 7.8 %), which suggested some demethylation had occurred. Furthermore, methylation patterns also varied between the two ovaries. Distinct patterns of DNA methylation arising through demethylation or de novo methylation might have specialized functions. This suggested the significance of epigenetic function in the development of orchid ovaries. The hypothesis that DNA methylation played a role in the ovary development of

C. hybridium

will help to illuminate the methylation status of given genes, to clone the fragments with different methylation patterns, and further shed novel insights into the molecular mechanisms of the ovary development of orchids from the point of view of epigenetics.

Xiaoqiang Chen, Xiulan Li, Ning Sun, Wenqin Song

Progress of Biotechnology

Frontmatter
Chapter 64. The Application Status of Microbes in Salted Fish Processing

Salted fish is a traditional Chinese aquatic product, because of its rich nutrition and the salty, sweet, unique taste and flavor, it is loved by consumers. At present, empirical traditional processing methods are adopted in enterprises that processing salted aquatic products, which have many disadvantages not only in the production scale, but also its efficiency. In order to innovate the traditional salted fish processing technology, as well as to meet the demand of modern consumers for salted fish with lower salt, richer nutrition, higher safety, better quality and flavor, this paper reviews researches on the isolation of dominant microorganisms in traditional salted fish and the application of lactic acid bacteria and other microorganisms in the salted fish processing, further, puts forward the tendency of applying modern microbial technologies to the processing of salted fish products.

Yan Yan Wu, Gang You, Lai Hao Li
Chapter 65. Construction and Functional Analysis of Luciferase Reporter Plasmids Containing ATM and ATR Gene Promoters

In eukaryotic cells, maintenance of genomic stability relies on the coordinated action of a network of cellular processes, including DNA replication, DNA repair, cell-cycle progression, and others. The DNA damage response (DDR) signaling pathway mediated by the ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) kinases is the central regulator of this network in response to DNA damage. The serine/threonine kinases ATM and ATR are the main kinases activated following various assaults on DNA. In this study, human ATM and ATR promoter luciferase reporter constructs were generated by PCR amplification. Then both PCR fragments respectively were digested and cloned into pGL3 vector. Finally, these promoter sequences were verified by sequencing. These results showed that luciferase reporter with ATM and ATR promoters were successfully constructed. Then the activation of the ATM promoter and ATR promoter following UV light treatments were detected in A431 cells by luciferase reporter assays. The results showed that UV damage could enhance transcriptional activity of ATM/ATR. Our research will provide useful tools for further deciphering ATM/ATR signaling and the pathways mediating the DNA damage response.

Li Zheng, Xing-Hua Liao, Nan Wang, Hao Zhou, Wen-Jian Ma, Tong-Cun Zhang
Metadata
Title
Advances in Applied Biotechnology
Editors
Tong-Cun Zhang
Motowo Nakajima
Copyright Year
2015
Publisher
Springer Berlin Heidelberg
Electronic ISBN
978-3-662-45657-6
Print ISBN
978-3-662-45656-9
DOI
https://doi.org/10.1007/978-3-662-45657-6

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