**8. References**

[1] Sukara E, Kumagai H, Yamamoto K. Study on amyloglucosidase of a newly isolated *Saccharomycopsis* sp. TJ-1 from the Indonesian fermented food (tape). *Annal Bogor*. 1998; 5 (2):77-83.

[2] Zhenming C, Chi Z, Liu G, Wang F, Ju L, Zhang T. *Saccharomycopsis fibuligera* and its applications in biotechnology. *Biotechnol Adv*. 2009; 27:423-431.

290 Chromatography – The Most Versatile Method of Chemical Analysis

works presented in this chapter were carried out.

*Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran,* 

*Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran,* 

*Biochemistry Research Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi* 

This paper is dedicated to Prof. Oei Ban Liang (1930-2010), the founder of the Inter-University Centre for Biotechnology, Bandung Institute of Technology, where most of the

The work has been supported by Indonesian Ministry of Research and Technology (RUT 1993-1996 to S.S), Indonesian Ministry of National Education (HB 2000-2003 to W.T.I), Royal Netherlands Academy of Arts and Science (KNAW SPIN mobility program 2004 to S.S), Indonesian Toray Science Foundation (STRG 2006 to K.H.), and Institut Teknologi Bandung (RU-ITB 2007-present to D.N.). We thank Prof. J.J. Beintema (Rijks*universiteit* Groningen) for

AEX, anion exchange chromatography; AMY, α-amylase from *S. fibuligera* R64; CD, cyclodextrin; CEX, cation exchange chromatography; DEAE, diethyl amino ethyl; DNA, deoxyribose nucleic acid; EDTA, ethylene diamine tetra acetate; FPLC, fast protein liquid chromatography; GLA, glucoamylase from *S. fibuligera* KZ; GLL1, glucoamylase from *S. fibuligera* R64; GLU, glucoamylase from *S. fibuligera* HUT7212; HCA, hydrophobic cluster analysis; HIC, hydrophobic interaction chromatography; LC, liquid chromatography; PDB, Protein Data Bank; PGK, phosphoglycerate kinase; RP-HPLC, reversed-phase high performance liquid chromatography; SDS PAGE, sodium dodecyl-sulfate polyacrylamide gel electrophoresis; SEC, size-exclusion chromatography; TFA, trifluoroacetic acid; TLC,

[1] Sukara E, Kumagai H, Yamamoto K. Study on amyloglucosidase of a newly isolated *Saccharomycopsis* sp. TJ-1 from the Indonesian fermented food (tape). *Annal Bogor*. 1998;

thin layer chromatography; TPCK, tosyl phenylalanyl chloromethyl ketone.

*Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compound in the Environment, Faculty of Science, Masaryk University, Brno, Czech Republic* 

Khomaini Hasan

Dessy Natalia

*Jatinangor, Sumedang, Indonesia* 

*Jatinangor, Sumedang, Indonesia* 

*Bandung, Bandung, Indonesia* 

valuable advice and discussion.

**Abbreviations** 

**8. References** 

5 (2):77-83.

**Acknowledgement** 

Toto Subroto and Soetijoso Soemitro


	- [20] Fuwa H. A new method for microdetermination of amylase activity by the use of amylose as the substrate. *J Biochem (Tokyo)*. 1954; 41:583-603.

Chromatography as the Major Tool in the Identification and

the Structure-Function Relationship Study of Amylolytic Enzymes from Saccharomycopsis Fibuligera R64 293

[39] Callebaut I, Labesse G, Durand P, Poupon A, Canard L, Chomilier J, et al. Deciphering protein sequence information through hydrophobic cluster analysis (HCA): current

[40] Raimbaud E, Bullion A, Perez S, Henrissat B. Hydrophobic cluster analysis of the primary sequences of α-amylases. *Int J Biol Macromol*. 1989; 11 (August):217-225. [41] Gaboriaud C, Bissery V, Benchetrit T, Mornon JP. Hydrophobic cluster analysis: an efficient new way to compare and analyse amino acid sequences. *FEBS Lett*. 1987; 224

[43] Ševčík J, Hostinová E, Solovicová A, Gašperík J, Dauter Z, Wilson KS. Structure of the complex of a yeast glucoamylase with acarbose reveals the presence of a raw starch

[44] Matsui I, Yoneda S, Ishikawa K, Miyairi S, Fukui S, Umeyama H, et al. Roles of the aromatic residues conserved in the active center of *Saccharomycopsis fibuligera* alphaamylase for transglycosylation and hydrolysis activity. *Biochem*. 1994; 33:189-196. [45] Sahdev S, Khattar S, Saini K. Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. *Mol Cell Biochem*.

[46] Matsui I, Matsui E, Ishikawa K, Miyairi S, Honda K. The enzymatic and molecular characteristics of *Saccharomycopsis* α-amylase secreted from *Saccharomyces cerevisiae*.

[47] Qiu X, Janson CA. Improving protein crystallizability by modifications and engineering. In: Bergfors TE, editor. Protein Crystallization. 2nd ed. La Jolla, CA, USA:

[48] Romanos MA, Scorer CA, Clare JJ. Foreign gene expression in yeast: a review. *Yeast*.

[49] Yamashita I, Itoh T, Fukui S. Cloning and expression of the *Saccharomycopsis fibuligera* glucoamylase gene in *Saccharomyces cerevisiae*. *Appl Microbiol Biotechnol*. 1985; 23 (2):130-

[50] Yamashita I, Itoh T, Fukui S. Cloning and expression of the *Saccharomycopsis fibuligera* alpha-amylase gene in *Saccharomyces cerevisiae*. *Agric Biol Chem*. 1985; 10:3089-3091. [51] Eksteen JM, van Rensburg P, Cordero Otero RR, Pretorius IS. Starch fermentation by recombinant *Saccharomyces cerevisiae* strains expressing the α-amylase and glucoamylase genes from *lipomyces kononenkoae* and *Saccharomycopsis fibuligera*. *Biotechnol Bioeng*. 2003;

[52] Subbaramiah K, Sharma R. Affinity purification of amylases on cyclodextrin-sepharose

[53] Hamilton LM, Kelly CT, Fogarty WM. Review: cyclodextrins and their interaction with

[54] Sauer Jr, Sigurskjold BW, Christensen U, Frandsen TP, Mirgorodskaya E, Harrison M, et al. Glucoamylase: structure/function relationships, and protein engineering. *Biochim* 

amylolytic enzymes. *Enzyme Microb Tech*. 2000; 26 (8):561-567.

*Biophys Acta - Prot Struct Mol Enzymol*. 2000; 1543 (2):275-293.

status and perspectives. *Cell Mol Life Sci*. 1997; 53:621-645.

[42] Ulmer KM. Protein Engineering. *Science*. 1983; 219 (4585):666-671.

binding site on the catalytic domain. *FEBS J*. 2006; 273 (10):2161-2171.

(1):149-155.

2008; 307 (1):249-264.

1992; 8:423-488.

84 (6):639-646.

133.

*Agric Biol Chem*. 1990; 54 (8):2009-2015.

International University Line; 2009. p.

columns. *Stärke*. 1989; 41 (9):357-359.


148.

(6):317-322.

submitted.

*FEBS Lett*. 1992; 304:1-3.

citrate buffers. *J Chem Soc*. 1953:4134-4136.

protein. *J Mol Biol*. 1982; 157 (1):105-132.

2001:E1.1.3-E.1.1.4.

amylose as the substrate. *J Biochem (Tokyo)*. 1954; 41:583-603. [21] Somogyi M. Notes on sugar determination. *J Biol Chem*. 1952; 195:19-23.

protocols. Heidelberg, Germany: Springer; 2005. p. 227-240.

mash saccharification. *Biotechnol Lett*. 1993; 15 (3):277-282.

*Songklanakarin J Sci Technol*. 2008; 30 ((supp. 1)):65-71.

and gel-electrophoresis. *Fresen J Anal Chem*. 1980; 301 (2):144-144.

enzymes of *Saccharomycopsis fibuligera*. *Int J Biochem*. 1991; 23 (1):21-25.

and peptides on spheron P-300. *J Chromatogr A*. 1978; 156 (2):239-254.

[20] Fuwa H. A new method for microdetermination of amylase activity by the use of

[22] Gasperik J, Kovac L, Minarikova O. Purification and characterization of the amylolytic

[23] Strop P, Mikes F, Chytilova Z. Hydrophobic interaction chromatography of proteins

[24] Webster J, Oxley D. Peptide Mass Fingerprinting. Chemical genomics: reviews and

[25] Rittinghaus K, Franzen KH. HPLC in protein analysis: an alternative to gel-filtration

[26] Soedjanaatmadja UMS, Hofsteenge J, Jeronimus-Stratingh CM, Bruins AP, Beintema JJ. Demonstration by mass spectrometry that pseudo-hevein and hevein have ragged Cterminal sequences. *Biochim Biophys Acta - Prot Struct Mol Enzymol*. 1994; 1209 (1):144-

[27] Walter A. Chymotrypsin: Molecular and catalytic properties. *Clin Biochem*. 1986; 19

[28] Tarr GE, Crabb JW. Reverse-Phase High-Performance Liquid Chromatography of

[29] Fourier E. Colorimetric quantification of carbohydrate. *Curr Protoc Food Anal Chem*.

[30] Ramesh MV, Lonsane BK. End product profiles of starch hydrolysis by bacterial alphaamylases at different temperature and pH values. *Biotech Lett*. 1989; 11 (9):649-652. [31] Joutsjoki VV, Parkkinen EEM, Torkkeli TK. A novel glucoamylase preparation for grain

[32] Saelim K, Dissara Y, H-Kittikun A. Saccharification of cassava starch by *Saccharomycopsis fibuligera* YCY1 isolated from Loog-Pang (rice cake starter).

[33] Ismaya WT, Hasan K, Kardi I, Zainuri A, Rahmawaty RI, Permanahadi S, et al. Chemical modification of *Saccharomycopsis fibuligera* R64 alpha-amylase to improve its stability against thermal, chelator, and proteolytic inactivation. *Appl Biochem Biotech*.

[34] Itoh T, Yamashita I, Fukui S. Nucleotide-sequence of the alpha-amylase gene (Alp1) in

[35] Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, et al. Protein identification and analysis tools on the ExPASy server. In: Walker JM, editor. The

[36] Janecek S, Balaz S. Alpha-amylases and approaches leading to their enhanced stability.

[37] Davies CW, Hoyle BE. 842. The Interaction of calcium ions with some phosphate and

[38] Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a

proteomics protocols handbook. New York: Humana Press; 2005. p. 571-607.

the yeast *Saccharomycopsis fibuligera*. *FEBS Lett*. 1987; 219 (2):339-342.

hydrophobic proteins and fragments thereof. *Anal Biochem*. 1983; 131:99-107.

	- [55] Gilkes NR, Kilburn DG, Miller RC, Warren RAJ. Structural and functional-analysis of a bacterial cellulase by proteolysis. *J Biol Chem*. 1989; 264 (30):17802-17808.

**Chapter 12** 

© 2012 Prabha and Kaur, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Prabha and Kaur, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Isolation and Purification of Sperm** 

**from Human Spermatozoa** 

Additional information is available at the end of the chapter

remain in the seminal fluid outside the body.

Vijay Prabha and Siftjit Kaur

http://dx.doi.org/10.5772/48594

**1. Introduction** 

**Immobilizing/Agglutinating Factors from** 

**Bacteria and Their Corresponding Receptors** 

For successful fertilization, motility is the most obvious and most essential sperm function and has been repeatedly shown to be predictive of fertilization in vitro [1]. Several studies have shown that the motility characteristics of spermatozoa are of the utmost importance for the men's fertility [2]. Spermatozoa dysfunction is the single most important cause of infertility. A decrease in spermatozoa motility with time is universal phenomenon. This reduction differs from species to species and also among individuals of the same species, as in the human male. Most investigators agree that the majority of spermatozoa cease to move within the first 24 hours. The survival of spermatozoa after ejaculation is dependent on the environmental conditions under which they are kept. In the female genital tract they may remain active for several days [3], but their activity is of much shorter duration if they

Male genital infections are relevant cause in the etiology of infertility due to abnormalities in sperm quality [4,5], affecting spermatozoal motility. The comparison of semen characteristics between infected and non-infected men show that motile spermatozoa are lower when the microorganisms are present in the semen [6]. It appears that bacteria have a direct effect on sperm motility with negative consequences in fertility. Among bacterial species that interact with spermatozoa are well-known causative pathogens of genitourinary infections such as *Escherichia coli, Ureaplasma urealyticum, Mycoplasma hominis, Chlamydia trachomatis* [7]. Of the various bacteria, *E. coli* is the most extensively studied microorganism in relation to infertility as a result of interaction with spermatozoa [8]. It is also the primary bacteria associated with prostatitis and epididymitis [9]. Several authors describe sperm

