Alkaliphiles

Koki Horikoshi

• Editore: Routledge
• Anno: 2021
• ISBN:
  9781351467445
• Edizione: 1
• Argomento: Medicina
• Categoria: Bioquímica en medicina

One of the most fascinating aspects of alkaliphiles is their ability to maintain pH homeostasis under extreme environmental conditions. This work provides a treatment of alkaliphilic microbiology, supported by molecular studies on the genetics of alkaliphilic "Bacillus" strain. Genomic analysis of "Bacillus halodurans" C-125 has been started and the genes responsible for alkaliphily are described. In addition to a basic background of alkaliphiles, including discussions of cell structures, physiology and molecular biology, "Alkaliphiles" presents an analysis of extracellular enzymes. Research on numerous enzymes including alkaline proteases, starch-degrading enzymes, cellulases, mannan-degrading enzymes, and many others is described in depth with relevant industrial applications.

7 Extracellular Enzymes

7.1 Isolation Procedures for Alkaline Enzymes

Enzymes having pH optimum for enzyme action in the alkaline regions are called alkaline enzymes. These include, for example, alkaline proteases (pH 10-12), alkaline amylases (pH 9-11), alkaline cellulases (pH 8-10), and others. Except for alkaline proteases these enzymes had not been isolated before our rediscovery of alkaliphilic microorganisms in 1968. Isolation of alkaline enzymes means the isolation of alkaliphilic microorganisms producing alkaline enzymes. Many screening procedures for these microorganisms have been developed. General procedures are described below with specific methods given in subsequent sections.

7.1.1 Protease Activity

Polypeptone in Horikoshi-I or -II medium is substituted for crude casein such as skim milk (1% w/v) and microorganisms are inoculated on the solidified media. After two or three days incubation at 37 °C, peptonization activity is directly observed as colonies hydrolyzing casein surrounded by a white halo. Candidates producing protease are cultured in the liquid media described in Table 7.1, and activity in the culture media are determined by conventional methods. Semi-quantitative assay using pulp disks is also a good method.

Table 7.1 Media for large-scale production

Ingredients (g/l)	Protease		Amylase				Cellulase	β-1,3-Glucanase
Soluble starch	50	40		15	80	80
CMC							20
Pachyman								20
Soya meal	20	30	30	20
Ground barley	50	100	100
Soya bean extract					10	10
Polypeptone					10	20	5	5
Fish extract						20
Na-caseinate	10
Soya bean oil		5.5
Polyglycol	0.1	0.1
Yeast extract							5	5
KH2PO4								1
Na2HPO4•10H2O	9			9
NaCl						2.5
CaCl2				1
CaCO3		5
MgSO4						0.2		0.2
Na2CO3	10	10			10	10	10	10
NaHCO3				10

7.1.2 Amylase Activity

Isolated microorganisms are grown in solidified Horikoshi-II medium. After two to three days incubation at 37 °C, iodine solution is poured onto plates. The colonies surrounded by a white halo are the amylase producers.

7.1.3 Cellulase Activity

Instead of glucose, 1% CMC is added to the Horikoshi-I medium solidified by agar containing 0.03% w/v Congo red, and soil samples spread on the plates. After two to three days incubation at 35-40 °C, white halos form around bacterial colonies producing CMCase.

7.1.4 β-1,3-Glucanase

Defatted pachyman powder is used as the β-1,3-glucan. Laminaran is also a good substrate for isolating microorganisms. Congo red (about 0.01-0.03% w/v) is added to the

media described above to indicate β-1,3-glucanase activity. The colonies surrounded by white halos are the enzyme producers. Pachyman should be defatted by ether, otherwise very poor growth of microorganisms is observed due to the presence of inhibitory substances in pachyman. Fig. 7.1 shows the media isolating the enzyme-producing microor-ganisms described above.

Fig. 7.1 An alkaliphilic Bacillus strain producing β-1,3-glucanase on congo red plate.

7.2 Alkaline Proteases

Alkaline proteases are not produced only by alkaliphilic microorganisms; two of the most widely studied enzymes are subtilisins BPN’ and Carlsberg produced by neutrophilic strains of Bacillus subtilis. They are not inhibited by metal chelators or thiol reagents; the N-terminal amino acid of both enzymes was identified as alanine and both were inhibited by a serine agent such as diisopropylfluorophosphate (DFP). Several alkaliphilic Streptomyces strains and Bacillus strains produce alkaline proteases capable of hydrolyzing keratinous proteins such as wool, hair, feather and silk under alkaline conditions (in 0.1N NaOH).

7.2.1 Alkaline Protease of Alkaliphilic Bacillus Strains

In 1971 Horikoshi (1971a) reported the production of an extracellular alkaline serine protease from alkaliphilk Bacillus sp. No. 221. This strain, isolated from soil, was found to produce large amounts of alkaline protease which differed from the subtilisin group. The enzyme was purified by DEAE and CM cellulose column chromatography and crystallized from ammonium sulfate solution. The optimum pH of the enzyme was 11.5 with 75% of the activity maintained at pH 13.0 (Fig. 7.2). The enzyme was completely inhibited by DFP or 6 M urea, but not by EDTA or p-chloromercuribenzoate. The molecular weight of the enzyme was 30,000, which is slightly higher than those of other reported alkaline proteases. Calcium ions affected both activity and stability of the enzyme. ...