Increase in hyaluronic acid production by Streptococcus equi subsp. zooepidemicus strain deficient in β-glucuronidase in laboratory conditions

Abstract

Streptococcus equi subsp. zooepidemicus is known to produce a hyaluronic acid capsule to resist the host immune defense. As the structure of the polysaccharide is identical to the one produced by humans, the bacteria S. equisubsp. zooepidemicusis used in biotechnological production of hyaluronic acid. In our laboratory we prepared mutated strains that are β-glucuronidase deficient. Comparing the wild-type strain, which is positive in β-glucuronidase activity, with the mutated strains named clone1 and clone2 in laboratory conditions, we observed that β-glucuronidase influences the production of hyaluronic acid considerably and the molecular weight of hyaluronan slightly. The production of hyaluronic acid by the mutated strains is higher by approximately 20% and the molecular weight is larger by about 2%. The significant increase in the production of hyaluronic acid and the slight increase in the molecular weight are probably caused by an absence of free β-glucuronic acid, due to its removal from the non-reducing termini of the polysaccharide by β-glucuronidase. The presence of free β-glucuronic acid would likely induce the expression of the β-glucuronic-acid-utilizing operon, which in turn would reflect into a misuse of energy in the glucose-rich media.

Appendices

Appendix A

First day: clone1
Cultivation time (h)	MW (Da)	Concentration (mg/ml)	Cell number	pH
6				7.31
8	1.969×106	0.1152	2.28×108	6.48
	1.963×106	0.1085
10	2.323×106	0.1699	3.74×108	5.1
	2.384×106	0.1594
12			4.53×108	4.95
	2.274×106	0.2357
14	2.240×106	0.2964	4.93×108	4.95
	2.110×106	0.3029
16	2.305×106	0.3255	5.28×108	5.15
	2.213×106	0.3423
18	2.239×106	0.3696	5.26×108	5.18
20	2.257×106	0.4184	5.49×108	5.25
	2.221×106	0.4247

Appendix B

First day: clone2
Cultivation time (h)	MW (Da)	Concentration (mg/ml)	Cell number	pH
6				7.3
8	2.493×106	0.0889	2.47×108	6.25
	2.318×106	0.0924
10	2.367×106	0.1716	3.86×108	4.9
	2.374×106	0.1679
12			4.63×108	4.9
	2.394×106	0.2184
14	2.264×106	0.2895	5.03×108	4.95
	2.343×106	0.2756
16	2.372×106	0.3033	5.29×108	5.05
	2.268×106	0.3151
18	2.289×106	0.3499	5.37×108	5.24
	2.190×106	0.3641
20	2.239×106	0.4111	5.44×108	5.24
	2.244×106	0.4065

Appendix C

First day: wild type
Cultivation time (h)	MW (Da)	Concentration (mg/ml)	Cell number	pH
6				7.48
8	2.570×106	0.0396	1.21×108	6.98
	2.611×106	0.0373
10	2.441×106	0.1173	2.85×108	5.4
	2.460×106	0.1147
12	2.253×106	0.1960	4.00×108	4.75
	2.136×106	0.2092
14	2.209×106	0.2505	4.41×108	4.84
	2.341×106	0.2329
16	2.386×106	0.2662	4.82×108	4.9
	2.242×106	0.2805
18	2.220×106	0.3208	4.69×108	4.88
	2.361×106	0.3020
20	2.282×106	0.3387	4.84×108	4.86
	2.275×106	0.3392

Appendix D

Second day: clone1
Cultivation time (h)	MW (Da)	Concentration (mg/ml)	Cell number	pH
6				5.36
8	2.461×106	0.1740	3.97×108	4.98
	2.457×106	0.1708
10	2.284×106	0.2453	4.69×108	4.97
	2.293×106	0.2461
12	2.256×106	0.2901	5.06×108	5.08
	2.384×106	0.2728
14	2.298×106	0.3203	5.28×108	5.28
	2.226×106	0.3269
16	2.193×106	0.3869	5.35×108	5.41
	2.284×106	0.3675
18	2.264×106	0.4048	5.47×108	5.3
	2.178×106	0.4281
20	2.204×106	0.4452	5.44×108	5.78
	2.184×106	0.4501

Appendix E

Second day: clone2
Cultivation time (h)	MW (Da)	Concentration (mg/ml)	Cell number	pH
6				5.36
8	2.497×106	0.1735	3.98×108	4.9
	2.347×106	0.1837
10	2.357×106	0.2402	4.71×108	4.96
	2.337×106	0.2397
12	2.209×106	0.3036	5.14×108	5.05
	2.260×106	0.2939
14	2.260×106	0.3359	5.29×108	5.26
	2.265×106	0.3342
16	2.271×106	0.3722	5.51×108	5.47
	2.295×106	0.3665
18	2.131×106	0.4354	5.60×108	5.33
	2.176×106	0.4304
20	2.231×106	0.4678	5.54×108	5.7
	2.178×106	0.4724

Appendix F

Second day: wild type
Cultivation time (h)	MW (Da)	Concentration (mg/ml)	Cell number	pH
6				5.28
8	2.483×106	0.1455	3.95×108	4.84
	2.380×106	0.1447
10	2.264×106	0.2124	4.53×108	4.8
	2.222×106	0.2150
12	2.188×106	0.2567	4.78×108	4.83
	2.239×106	0.2528
14	2.159×106	0.2814	4.89×108	4.87
	2.179×106	0.2781
16	2.006×106	0.3315	5.11×108	5.07
	2.170×106	0.3119
18	2.104×106	0.3390	5.13×108	5.05
	2.170×106	0.3239
20	2.133×106	0.3576	5.13×108	5.27
	2.025×106	0.3692

Appendix G

Third day: clone1
Cultivation time (h)	MW (Da)	Concentration (mg/ml)	Cell number	pH
6				6.06
8	2.378×106	0.1714	3.77×108	5.00
	2.391×106	0.1696
10	2.294×106	0.2393	4.50×108	5.00
	2.224×106	0.2384
12	2.154×106	0.3148	4.97×108	5.05
	2.287×106	0.3044
14	2.285×106	0.3230	5.20×108	5.15
	2.299×106	0.3247
16	2.293×106	0.3644	5.27×108	5.34
	2.261×106	0.3772
18	2.199×106	0.4445	5.45×108	5.45
	2.382×106	0.4074
20	2.354×106	0.4520	5.54×108	5.54
	2.200×106	0.4677

Appendix H

Third day: clone2
Cultivation time (h)	MW (Da)	Concentration (mg/ml)	Cell number	pH
6				5.58
8	2.289×106	0.1769	3.90×108	4.94
	2.336×106	0.1710
10	2.244×106	0.2407	4.62×108	5.00
	2.222×106	0.2420
12	2.354×106	0.2662	4.98×108	5.10
	2.392×106	0.2712
14	2.144×106	0.3344	5.15×108	5.23
	2.185×106	0.3292
16	2.188×106	0.3572	5.26×108	5.45
	2.168×106	0.3548
18	2.254×106	0.4158	5.35×108	5.55
	2.248×106	0.4147
20	2.413×106	0.4262	5.43×108	5.68
	2.351×106	0.4264

Appendix I

Third day: wild type
Cultivation time (h)	MW (Da)	Concentration (mg/ml)	Cell number	pH
6				6.46
8	2.425×106	0.1457	3.32×108	5.09
	2.456×106	0.1446
10	2.329×106	0.2064	4.14×108	4.85
12	2.256×106	0.2421	4.45×108	4.95
	2.250×106	0.2438
14	2.285×106	0.2562	4.47×108	4.87
	2.186×106	0.2780
16	2.190×106	0.3043	4.52×108	4.95
	2.227×106	0.2865
18	2.268×106	0.3219	4.62×108	4.96
	2.193×106	0.3205
20	2.233×106	0.3411	4.72×108	5.15
	2.245×106	0.3229

Appendix J

Fourth day: clone1
Cultivation time (h)	MW (Da)	Concentration (mg/ml)	Cell number	pH
6				6.85
8	2.168×106	0.1532	3.19×108	5.16
10	2.145×106	0.2205	4.26×108	4.96
12	2.143×106	0.2785	4.74×108	4.93
	2.190×106	0.2705
14	2.210×106	0.2835	5.03×108	5.17
	2.197×106	0.2908
16	2.297×106	0.3370	5.19×108	5.25
	2.293×106	0.3389
18	2.215×106	0.4039	5.32×108	5.4
	2.250×106	0.3885
20	2.243×106	0.4236	5.38×108	5.94

Appendix K

Fourth day: clone2
Cultivation time (h)	MW (Da)	Concentration (mg/ml)	Cell number	pH
6				6.31
8	2.260×106	0.1873	3.96×108	4.85
	2.276×106	0.1824
10	2.354×106	0.2402	4.84×108	5.00
	2.241×106	0.2569
12	2.354×106	0.3103	5.23×108	4.86
	2.243×106	0.3168
14	2.130×106	0.3604	5.46×108	5.16
	2.239×106	0.3559
16	2.176×106	0.3945	5.63×108	5.20
	2.108×106	0.4123
18	2.354×106	0.4386	5.75×108	5.40
	2.240×106	0.4469
20	2.223×106	0.4834	5.80×108	5.97
	2.221×106	0.4804

Appendix L

Fourth day: wild type
Cultivation time (h)	MW (Da)	Concentration (mg/ml)	Cell number	pH
6				5.94
8	2.178×106	0.1870	3.91×108	4.85
	2.279×106	0.1831
10	2.253×106	0.2574	4.63×108	4.90
	2.159×106	0.2639
12	2.295×106	0.2747	4.82×108	4.85
14	2.121×106	0.3252	4.94×108	4.90
	2.118×106	0.3245
16	2.064×106	0.3520	5.00×108	4.93
	2.156×106	0.3370
18	2.258×106	0.3406	4.99×108	5.04
	2.258×106	0.3383
20	2.247×106	0.3422	5.08×108	5.42
	2.196×106	0.3462